Glutaminase inhibitor therapy

ABSTRACT

Disclosed herein are methods of treating a tumor or cancer in a subject whose tumor or cancer cells express low levels of asparagine synthetase (ASNS), and compounds and compositions useful in such treatment. Also disclosed herein are methods of evaluating whether to administer a compound that inhibits glutathione production or a glutaminase inhibitor to a subject with a tumor or cancer.

This application is a continuation of U.S. patent application Ser. No.16/164,581, filed Oct. 18, 2018, which application issued on Jul. 28,2020 as U.S. Pat. No. 10,722,487, which claims the benefit of priorityof U.S. Provisional Application No. 62/573,906, filed Oct. 18, 2017, thedisclosures of which are hereby incorporated by reference as if writtenherein in their entireties.

Metabolic deregulation is a hallmark of cancer as tumors exhibit anincreased demand for nutrients and macromolecules to fuel their rapidproliferation. Glutamine (Gln), the most abundant amino acid incirculation, plays an essential role in providing cancer cells withbiosynthetic intermediates required to support proliferation andsurvival. Specifically, tumor cells utilize glutaminolysis, or theenzymatic conversion of glutamine to glutamate, as a nutrient source foramino acid and nucleotide synthesis, and a carbon skeleton to fuel ATPand NADPH synthesis through the TCA cycle.

In addition to enabling cell growth and replication, glutaminemetabolism plays a critical role in multiple metabolic events togenerate energy and maintain cellular redox balance. Products ofglutaminolysis are used in maintaining cellular redox homeostasis asglutamate can be converted into glutathione, the major intracellularantioxidant. Cancer cells require a constant source of biomass andmacro-molecules to support cell division and reducing agents to maintainredox homeostasis. While many of these building blocks are providedthrough aerobic glycolysis, many cancer cells have evolved a dependenceon glutamine metabolism for growth and survival.

Glutamine metabolism, i.e., glutaminolysis is regulated by mitochondrialglutaminase (GLS), the rate limiting enzyme that catalyzes theconversion of glutamine to glutamate and ammonia. Mammalian cellscontain two genes that encode glutaminase: the kidney-type (GLS-1) andliver-type (GLS-2) enzymes. Each has been detected in multiple tissuetypes, with GLS-1 being widely distributed throughout the body. GLS-1 isa phosphate-activated enzyme that exists in humans as two major splicevariants, a long form (referred to as KGA) and a short form (GAC), whichdiffer only in their C-terminal sequences. Both forms of GLS-1 arethought to bind to the inner membrane of the mitochondrion in mammaliancells, although at least one report suggests that glutaminase may existin the intramembrane space, dissociated from the membrane. GLS isfrequently overexpressed in human tumors and has been shown to bepositively regulated by oncogenes such as Myc. Consistent with theobserved dependence of cancer cell lines on glutamine metabolism,pharmacological inhibition of GLS offers the potential to target Glnaddicted tumors. Such targeted treatment, however, is hampered by thelack of clinical biomarkers to identify sensitive patient populations.

Thus, there is a need for a means to identify markers and mechanismsthat provide a method for evaluating a patient's successful treatmentwith glutaminase inhibitors as well as for methods of such treatment.

The present invention is based, in part, on the discovery of a mechanismwhereby tumor cells with a specific metabolic dependence on the activityof glutaminase to generate intracellular pools of glutamate are uniquelysensitive to glutaminase inhibition. Conversely, cells which havealternative mechanisms for generating glutamate define a specificpatient population that may be insensitive to glutaminase inhibitors.

In one embodiment of the invention, the invention comprises a method oftreating cancer in a subject whose cancer cells express low levels ofasparagine synthetase (ASNS), as defined by an Histophathology Score(H-score) of 0-100 (both inclusive) by immunohistochemical staining,comprising administering a glutaminase-1 (GLS-1) inhibitor to saidsubject.

In another embodiment of the invention, the invention comprises a methodof treating a subject having a disorder, such as a cancer or tumor. Themethod comprises determining the concentration or expression of ASNS insaid cancer or tumor of said subject; and administering a glutaminase-1(GLS-1) inhibitor to said subject if the level of ASNS is quantified asan H-score of less than or equal to 100 by immunohistochemical staining.In another embodiment of the invention, the method comprises optionallyobtaining a biological sample from a subject, determining that theconcentration or expression of ASNS in said sample from said subject islow and administering a glutaminase-1 (GLS-1) inhibitor to said subject.

In yet another embodiment of the invention, the invention comprises amethod of stratifying a subject for response to GLS inhibitor therapy.The method comprises determining that the concentration of ASNS or itsexpression levels in a tumor or in cancer cells of said subject are lowand administering a glutaminase inhibitor to said subject.

The subject in need of treatment may be afflicted with a disorder or acondition, for example, cancer, including, but not limited to, bladdercancer, bone marrow cancer, breast cancer, cancer of the central nervoussystem, cervical cancer, colon cancer, endometrial cancer, cancer of thegastric system, head and neck cancer, kidney cancer, leukemia, livercancer, lung cancer, lymphoma, muscle cancer, ovarian cancer, pancreaticcancer, prostate cancer, skin cancer, thyroid cancer, or a variantthereof. In some embodiments, the cancer is ovarian, skin, liver,prostate, breast, colon, lung, head and neck cancers or a lymphoma. Insome embodiments, the cancer is ovarian cancer, for example, high-gradeserous ovarian cancer (HGSOC). The GLS-1 inhibitor may, for example, bea selective inhibitor of GLS-1.

BRIEF DESCRIPTION OF FIGURES

FIG. 1(a) shows the the potent and specific inhibition of glutaminase-1(GLS-1) by compound 1. x-axis=concentration of compound 1 (M);y-axis=activity (relative to control); IC₅₀=5.8 nm.

FIG. 1(b) shows that target engagement for OVCAR8 can be measured aftertreatment with compound 1. The glutamate:glutamine ratio is depicted forDMSO (1.) and Compound 1 (2.).

FIG. 1(c) shows that target engagement for OVCAR429 can be measuredafter treatment with compound 1. The glutamate:glutamine ratio isdepicted for DMSO (1.) and Compound 1 (2.).

FIG. 1(d) shows that target engagement for IGROV1 can be measured aftertreatment with compound 1. The glutamate:glutamine ratio is depicted forDMSO (1.) and Compound 1 (2.).

FIG. 1(e) shows that target engagement for OVCAR4 can be measured aftertreatment with compound 1. The glutamate:glutamine ratio is depicted forDMSO (1.) and Compound 1 (2.).

FIG. 2(a) shows that differential response to GLS-1 inhibition isobserved in OVCA lines. Viability (relative to DMSO) of OVCAR8 isplotted as a function of Compound 1 concentration (uM). IC₅₀=0.013 uM.

FIG. 2(b) shows that differential response to GLS-1 inhibition isobserved in OVCA lines. Viability (relative to DMSO) of OVCAR429 isplotted as a function of Compound 1 concentration (uM). IC₅₀=0.059 uM.

FIG. 3(a) shows that differential response to GLS-1 inhibition isobserved in OVCA lines. Viability (relative to DMSO) of IGROV1 isplotted as a function of Compound 1 (uM).

FIG. 3(b) shows that differential response to GLS-1 inhibition isobserved in OVCA lines. Viability (relative to DMSO) of OVCAR4 isplotted as a function of Compound 1 concentration (uM).

FIG. 4 shows a waterfall plot depicting differential response to GLS-1inhibition in a broad panel of OVCA lines. IC50 (uM) at 72 h is plottedfor several cell lines. A subset of OVCA lines show low nM sensitivityto GLSi while others do not show any response.

FIG. 5 shows that metabolic alterations result in an altered redoxbalance in response to treatment of OVCAR429 (1.) and OVCAR8 (2.) withcompound 1. Metabolite fold change (log 2) is plotted for variousmetabolites involved in: TCA (i), glutamate synthesis (ii); purine(iii); and pentose phosphate (iv) pathways.

FIG. 6 shows that metabolic alterations result in an altered redoxbalance in response to treatment of OVCAR4 (1.) and IGROV1 (2.) withcompound 1. Metabolite fold change (log 2) is plotted for variousmetabolites involved in: TCA (i), glutamate synthesis (ii); purine(iii); and pentose phosphate (iv) pathways.

The next six figures show that GLS-dependence is driven by addiction toglutamine-dependent, glutathione-mediated redox maintenance in OVCAresponder cell lines.

FIG. 7 shows that glutathione levels are decreased after treatment withcompound 1 (2.) compared to DMSO (1.). Shown are the glutathione levels(uM) for responders (i) OVCAR420 (a); OVCAR429 (b); OVCAR8 (c); and anon-responder (ii) OVCAR4 (d).

FIG. 8(a) shows that the loss of glutathione (GSH) after treatment withcompound 1, a GLS-1 inhibitor (GLS1i), leads to an accumulation ofintracellular reactive oxygen species (ROS) in OVCAR420 cells.

FIG. 8(b) shows that the loss of glutathione (GSH) after treatment withcompound 1, a GLS-1 inhibitor (GLS1i), leads to an accumulation ofintracellular reactive oxygen species (ROS) in OVCAR429 cells.

FIG. 9(a) shows a standard BrdU assay with OVCAR420 using DMSO control.x-axis=PI-A; y-axis=FITC-A.

FIG. 9(b) shows a standard BrdU assay with OVCAR420 using GLS inhibitor.x-axis=PI-A; y-axis=FITC-A.

FIG. 10(a) shows a standard BrdU assay with OVCAR429 using DMSO control.x-axis=PI-A; y-axis=FITC-A.

FIG. 10(b) shows a standard BrdU assay with OVCAR429 using GLSinhibitor. x-axis=PI-A; y-axis=FITC-A.

FIG. 11 shows that the GLS1i induces oxidative stress that leads toaccumulation of DNA damage. y-axis is yH2AX foci, normalized to DMSO.(a): OVCAR8; (b) OVCAR420. In both cases, data are shown for DMSO (1.)and Compound 1 (2.).

FIG. 12(a) shows that application of exogenous GSH to responder cellline OVCAR420 rescues proliferation defects induced by compound 1.Viability upon application of cell-permeable GSH (1.) is shown, alongwith a control (2) without GSH treatment.

FIG. 12(b) shows that application of exogenous GSH to responder cellline OVCAR429 rescues proliferation defects induced by compound 1.Viability upon application of cell-permeable GSH (1.) is shown, alongwith a control (2) without GSH treatment.

The next four figures show that asparagine synthetase (ASNS) expressionis a negative predictor of response after GLS-1 inhibition.

FIG. 13 shows key metabolic pathways that determine the production andfate of glutamine (GLU).

FIG. 14 shows the ASNS levels by reverse phase protein array (RPPA)expression analysis in responder (a) and non-responder (b) OVCA celllines.

FIG. 15 shows the Western blot of ASNS expression across a panel of OVCAcell lines. ASNS (1.); HSP90 (2.). Responder cell lines are enclosed inbrackets.

FIG. 16(a) shows western blot of ASNS for ASNS over-expression study.OV8 (1.); ASNS OE (2.); ASNS OE 1:10 (3.); ASNS OE (1:100).

FIG. 16(b) shows western blot of GAPDH for over-expression study. OV8(1.); ASNS OE (2.); ASNS OE 1:10 (3.); ASNS OE (1:100).

FIG. 16(c) plots viability, normalized to DMSO, for OV8 (1.); OV8 ASNSOE (2.); OV8 ASNS OE 1:10 (3.); and OV8 ASNS OE 1:100 (4.).

The next three figures show that immuno-histochemical staining for ASNSconfirms differential expression between responders and non-responders.

FIG. 17(a) shows ASNS western blot for ASNS knockdown study. NTC (1.)and ASNS KD (2.).

FIG. 17(b) shows Hsp90 western blot for ASNS knockdown study. NTC (1.)and ASNS KD (2.).

FIG. 17(c) (NTC) shows ASNS immunohistochemical (IHC) antibodyvalidation.

FIG. 17(d) (KD) shows ASNS immunohistochemical (IHC) antibodyvalidation.

FIG. 18(a) shows IHC assay for responder OVCAR420.

FIG. 18(b) shows IHC assay for responder OVCAR429.

FIG. 18(c) shows IHC assay for non-responder OVCAR4.

FIG. 18(d) shows IHC assay for non-responder A2780 (d).

FIG. 19(a) shows tissue microarrays from ovarian cancer patients withlow ASNS expressing TMA cores.

FIG. 19(b) shows tissue microarrays from ovarian cancer patients withhigh ASNS expressing TMA cores.

FIG. 19(c) shows provides quantification of patients with differentlevels of ASNS expression from ASNS scoring.

The next three figures show that glutaminase inhibition inhibits tumorgrowth in ASNS_(low) models of ovarian cancer.

FIG. 20(a) shows that OVCAR-8 subcutaneous xenografts are sensitive toGLS-1 inhibition. x-axis: day; y-axis=tumor volume (mm³).

FIG. 20(b) shows that GLS-1 inhibition inhibits tumor progression in anorthotopic model of ovarian cancer. Vehicle (1.); Compound 2 100 mpk(2.); Paclitaxel 15 mpk (3.); and Compound 2+Paclitaxel (4.);y-axis=combined nodule weight (g).

FIG. 21 shows that OVCAR-8 tumors demonstrate altered glutaminemetabolism after GLS-1 inhibition. x-axis=day;y-axis=glutamate:glutamine ratio. Vehicle (1.) and Compound 2 (2.).

FIG. 22(a) shows phospho-histone H3 stain for Compound 1.

FIG. 22(b) shows phospho-histone H3 stain for vehicle.

FIG. 22(c) shows pathologist score for vehicle (1.) and Compound 1 (2.).

The next two figures show that glutaminase inhibition inhibits tumorgrowth in an ASNS_(low) patient-derived xenograft (PDX) model of ovariancancer.

FIG. 23(a) shows scoring of patients samples using a traditionalpathologist's scale of 0-3.

FIG. 23(b) shows example stains of TMA cores having low ASNS expression.

FIG. 23(c) shows example stains of TMA cores having ASNS expression.

FIG. 24(a) shows a summary of scoring from TMA cores, which reveals 40%of patients with no or low ASNS expression, and another 20% with mediumlevels of ASNS expression. Both populations would be predicted torespond to GLS inhibition.

FIG. 24(b) shows that growth of an ASNS_(low) PDX model is inhibited invivo following treatment with GLSi. Vehicle (1.); Compound 2 100 mpk(2.); Paclitaxel 15 mpk (3.); and Compound 2+ Paclitaxel (4.).

FIG. 25(a) shows production of glutathione and subsequent reduction ofreactive oxygen species (ROS).

FIG. 25(b) shows that reduction of reactive oxygen species (ROS) levelscan be reduced in responders by treatment with a GLS inhibitor, thusincreasing ROS levels.

FIG. 25(c) shows that non-responders circumvent GLS inhibition byutilizing aspartate as an alternate feedstock for glutathione synthesis.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of biomarkersand mechanisms that indicate responsiveness to glutaminase (GLS)inhibitor treatment. Specifically, a molecular mechanism has beenidentified that stratifies cancer patients for treatment and indicatesresponsiveness to GLS inhibition. In particular, the inventors havediscovered that some cancer cells are sensitive to GLS inhibition. Theinventors have also discovered that cells that express high levels ofasparagine synthetase (ASNS) are resistant to inhibition of GLS.

Without being bound to any theory, it is believed that cells, forexample, tumor cells, which are sensitive to GLS-1 inhibition display adependence on reduced glutathione, the major endogenous antioxidantcomprised of glycine, cysteine, and glutamine-derived glutamate.Inhibition of GLS-1 reduces the steady state levels of glutathione, thusshifting the redox balance of such cells. Consistent with this model,treatment of a subset of HGSOC cell lines with a potent GLS-1 inhibitorreduces intracellular levels of glutamate and glutathione and inhibitscell growth through a mechanism that involves reactive oxygen species(ROS)-induced DNA damage.

In contrast, HGSOC cell lines expressing high levels of ASNS, the enzymethat metabolizes aspartate to glutamate and asparagine, failed torespond to GLS-1 inhibitor treatment. These non-responder cells maintainglutamate and glutathione pools in the presence of GLS-1 inhibitionthrough the activity of ASNS, and presumably utilize ASNS-derivedglutamate to produce glutathione. Consequently, ASNS levels function asa negative indicator of response to GLS-1 inhibition, thus, serving as apatient stratification biomarker.

Nearly 21,000 new ovarian cancer patients are diagnosed each year, andapproximately 14,000 deaths result from this disease in the UnitedStates alone. While surgical resection of these tumors is becoming moresuccessful, when tumors are inoperable, or when resection is incomplete,there are no effective treatments. This invention provides a mechanismto identify, and methods of treatment of, metabolically distinct subsetsof ovarian cancer patients that will respond to targeted GLS-1 inhibitor(GLS1i) therapy. Further, as ASNS expression levels indicate response toGLS-1 inhibition, the expression levels of this enzyme in cancerouscells serves as an invaluable marker for GLS1i therapy and the treatmentof ovarian cancer patients.

Accordingly, in one aspect of the invention, the invention comprises amethod of treating a tumor or a cancer in a subject. In one embodiment,the invention comprises a method of treating a tumor or a cancer in asubject wherein the tumor or cancer cells in said subject express lowlevels of asparagine synthetase (ASNS), comprising administering aglutaminase inhibitor to said subject.

In another embodiment of the invention, the invention comprises a methodof treating a subject having a disorder, such as a cancer or tumor. Themethod comprises determining that the concentration or expression ofASNS in said cancer or tumor of said subject is low and administering aglutaminase inhibitor to said subject.

Methods and techniques of determining whether the concentration orexpression of ASNS in the tumor or cancer cells of a subject are low ornot are known to one of skill in the art and are also described herein.Any method known to one of skill in the art can be used, including, butnot limited to, determining the Histophathology Score (H-score) of thetumor or cancer cells by immunohistochemical staining. An H-score ofless than or equal to 100 indicates that the level of ASNS in the tumoror cancer cells of the subject are low.

In another embodiment of the invention, the invention comprises treatingcancer in a subpopulation of subjects, the subpopulation beingcharacterized by a low level of ASNS in the subjects, and administeringa glutaminase inhibitor to the subjects in the subpopulation. In someembodiments, the level of ASNS in the subjects in the subpopulation isquantified as an H-score by immunohistochemical staining. In someembodiments, the H-score of subjects in the subpopulation is less thanor equal to 150. In some embodiments, the H-score of subjects in thesubpopulation is less than or equal to 125. In some embodiments, theH-score of subjects in the subpopulation is less than or equal to 100.

Embodiment 1: A method for treatment of a disorder in a subjectcharacterized by a low level of expression or concentration ofasparagine synthetase (ASNS), said treatment comprising administeringone or more glutathione lowering agents to said subject.

Embodiment 2: A method for treatment of a disorder in a subpopulation ofsubjects, said subjects in said subpopulation being characterized by alow level of expression or concentration of ASNS, said treatmentcomprising administering one or more glutathione lowering agents to saidsubjects.

Embodiment 3: A method of stratifying a subject for response to GLS-1inhibitor therapy, comprising determination of a low level of expressionor concentration of ASNS in said subject, and administering one or moreglutathione lowering agents to said subject.

Embodiment 4: A method comprising the treatment of a subject having adisorder in need of treatment, comprising determination of theexpression or concentration or asparagine synthetase (ASNS) in thesubject, and administering one or more glutathione lowering agents tosaid subject if said subject displays a low level of expression orconcentration of ASNS.

Embodiment 5: The method of any one of Embodiments 1-4, furthercomprising obtaining a biological sample or samples from said subject orsubjects.

Embodiment 6: The method of Embodiment 4, further comprising determiningthe level or concentration of ASNS from said biological sample orsamples.

Embodiment 7: The method of any one of Embodiments 1-6, wherein saiddisorder is chosen from a cancer or a tumor.

Embodiment 8: The method of Embodiment 6, wherein said disorder is acancer.

Embodiment 9: The method of Embodiment 8, wherein said cancer is chosenfrom Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma andLymphoma), Anal Cancer, Appendix Cancer, Atypical Teratoid/RhabdoidTumor, Basal Cell Carcinoma, Bile Duct Cancer (including Extrahepatic),Bladder Cancer, Bone Cancer (including Osteosarcoma and MalignantFibrous Histiocytoma), Brain Tumor (such as Astrocytomas, Brain andSpinal Cord Tumors, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors,Craniopharyngioma, Ependymoblastoma, Ependymoma, Medulloblastoma,Medulloepithelioma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, BasalCell Carcinoma, Bile Duct Cancer (including Extrahepatic), BladderCancer, Bone Cancer (including Osteosarcoma and Malignant FibrousHistiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary, CentralNervous System (such as Atypical Teratoid/Rhabdoid Tumor, EmbryonalTumors and Lymphoma), Cervical Cancer, Childhood Cancers, Chordoma,Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma (Mycosis Fungoides andSézary Syndrome), Duct, Bile (Extrahepatic), Ductal Carcinoma In Situ(DCIS), Embryonal Tumors (Central Nervous System), Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer(like Intraocular Melanoma, Retinoblastoma), Fibrous Histiocytoma ofBone (including Malignant and Osteosarcoma), Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalStromal Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia,Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors (Endocrine, Pancreas), KaposiSarcoma, Kidney (including Renal Cell), Langerhans Cell Histiocytosis,Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), AcuteMyeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML),Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSézary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer (such as Epithelial, GermCell Tumor, Low Malignant Potential Tumor and High Grade Serous OvarianCancer), Pancreatic Cancer (including Islet Cell Tumors),Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma,Pineal Parenchymal Tumors of Intermediate Differentiation, Pineoblastomaand Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor,Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma,Pregnancy and Breast Cancer, Primary Central Nervous System (CNS)Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer,Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma,Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (like Ewing SarcomaFamily of Tumors, Kaposi, Soft Tissue, Uterine), Sézary Syndrome, SkinCancer (such as Melanoma, Merkel Cell Carcinoma, Nonmelanoma), SmallCell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, SquamousCell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic,Stomach (Gastric) Cancer, Supratentorial Primitive NeuroectodermalTumors, T-Cell Lymphoma (Cutaneous, Mycosis Fungoides and SézarySyndrome), Testicular Cancer, Throat Cancer, Thymoma and ThymicCarcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvisand Ureter, Trophoblastic Tumor (Gestational), Unknown Primary, UnusualCancers of Childhood, Ureter and Renal Pelvis, Transitional Cell Cancer,Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma,Waldenström Macroglobulinemia and Wilms Tumor, or a variant thereof.

Embodiment 10: The method of Embodiment 8, wherein said cancer is chosenfrom high grade serous (HGSOC), epithelial, germ cell tumor, and lowmalignant potential tumor.

Embodiment 11: The method of Embodiment 8, wherein said cancer is chosenfrom ovarian, skin, liver, prostate, breast, colon, lung, head and neckcancers and lymphoma.

Embodiment 12: The method of Embodiment 11, wherein said cancer isovarian cancer.

Embodiment 13: The method of Embodiment 12, wherein said ovarian canceris high-grade serous ovarian cancer (HGSOC).

Embodiment 14: The method of Embodiment 13, wherein said ovarian canceris nonresectable or relapsed HGSOC.

Embodiment 15: The method of Embodiment 7, wherein said disorder is atumor or tumors.

Embodiment 16: The method of any one of Embodiments 1-15, wherein saidlow level of expression or concentration of ASNS is a low level ofexpression of ASNS.

Embodiment 17: The method of Embodiment 16, wherein said tumor or tumorsof said subject or subjects is characterized by a low level ofexpression of ASNS.

Embodiment 18: The method of any one of Embodiments 1-15, wherein saidlow level of expression or concentration of ASNS is a low concentrationof ASNS.

Embodiment 19: The method of Embodiment 16, wherein which said tumor ortumors of said subject or subjects is characterized by a lowconcentration of ASNS.

Embodiment 20: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 150 byimmunohistochemical staining.

Embodiment 21: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 125 byimmunohistochemical staining.

Embodiment 22: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 100 byimmunohistochemical staining.

Embodiment 23: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 95 byimmunohistochemical staining.

Embodiment 24: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 90 byimmunohistochemical staining.

Embodiment 25: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 85 byimmunohistochemical staining.

Embodiment 26: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 80 byimmunohistochemical staining.

Embodiment 27: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 75 byimmunohistochemical staining.

Embodiment 28: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 70 byimmunohistochemical staining.

Embodiment 29: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 65 byimmunohistochemical staining.

Embodiment 30: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 60 byimmunohistochemical staining.

Embodiment 31: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 55 byimmunohistochemical staining.

Embodiment 32: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 50 byimmunohistochemical staining.

Embodiment 33: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 45 byimmunohistochemical staining.

Embodiment 34: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 40 byimmunohistochemical staining.

Embodiment 35: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 35 byimmunohistochemical staining.

Embodiment 36: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 30 byimmunohistochemical staining.

Embodiment 37: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 25 byimmunohistochemical staining.

Embodiment 38: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 20 byimmunohistochemical staining.

Embodiment 39: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 15 byimmunohistochemical staining.

Embodiment 40: The method of either one of Embodiment 18 or 19, in whichsaid concentration of ASNS is less than or equal to 10 byimmunohistochemical staining.

Embodiment 41: The method of any one of Embodiments 1-40, wherein saidone or more glutathione lowering agents comprises one or more compoundsthat inhibit glutathione production or activity.

Embodiment 42: The method of Embodiment 41, wherein said one or moreglutathione lowering agents comprises one or more compounds that inhibitglutathione production.

Embodiment 43: The method of Embodiment 41, wherein said one or moreglutathione lowering agents comprises one or more compounds that inhibitglutathione activity.

Embodiment 44: The method of any one of Embodiments 1-43, wherein saidone or more glutathione lowering agents comprises one or more compoundsthat inhibits amino acid or glutathione transport.

Embodiment 45: The method of Embodiment 44, wherein said one or moreglutathione lowering agents comprises one or more compounds thatinhibits amino acid transport.

Embodiment 46: The method of Embodiment 44, wherein said one or moreglutathione lowering agents comprises one or more compounds thatinhibits glutathione transport.

Embodiment 47: The method of any one of Embodiments 1-46, wherein saidone or more glutathione lowering agents comprises one or moreglutaminase inhibitor.

Embodiment 48: The method of Embodiment 47, wherein said one or moreglutaminase inhibitor comprises one or more GLS-1 inhibitors.

Embodiment 49: The method of Embodiment 48, wherein said one or moreGLS-1 inhibitors comprises one or more selective GLS-1 inhibitors.

Embodiment 50: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises one or more GLS-1 inhibitorschosen from the group consisting of:

-   -   a)        (S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,    -   b)        N,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),        also known as BPTES,    -   c)        2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3-(trifluoromethoxy)phenyl]acetamido}pyridazin-3-yl)butyl]-1,3,4-thiadiazol-2-yl}acetamide,        also known as CB-839,    -   d)        N,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide),    -   e)        N-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)phenyl]pyridin-2-yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide,    -   f)        1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide,    -   g)        1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   h)        N-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide,    -   i)        (R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)phenyl)pyridin-2-yl)acetamido)-pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   j)        (R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   k)        (R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   1)        (R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   m)        (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   n)        (R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)-acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   o)        1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   p)        1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   q)        1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   r)        1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   s)        (R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   t)        5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide,        and    -   u)        N,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)        (both or either of 1S,3S and 1R,2R enantiomers), or a salt and        polymorph thereof.

Embodiment 51: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises two or more GLS-1 inhibitorschosen from the group consisting of:

-   -   a)        (S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,    -   b)        N,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),        also known as BPTES,    -   c)        2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3-(trifluoromethoxy)phenyl]acetamido}pyridazin-3-yl)butyl]-1,3,4-thiadiazol-2-yl}acetamide,        also known as CB-839,    -   d)        N,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide),    -   e)        N-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)phenyl]pyridin-2-yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide,    -   f)        1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide,    -   g)        1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   h)        N-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide,    -   i)        (R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)phenyl)pyridin-2-yl)acetamido)-pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   j)        (R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   k)        (R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   l)        (R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   m)        (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   n)        (R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)-acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   o)        1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   p)        1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   q)        1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   r)        1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   s)        (R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,    -   t)        5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide,        and    -   u)        N,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)        (both or either of 1S,3S and 1R,2R enantiomers), and/or salts        and polymorphs thereof.

Embodiment 52: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,or a salt or polymorph thereof.

Embodiment 53: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof.

Embodiment 54: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(S)-2-hydroxy-2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,or a salt or polymorph thereof.

Embodiment 55: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide),or a salt or polymorph thereof.

Embodiment 56: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)-phenyl]pyridin-2-yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 57: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 58: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 59: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3-(trifluoromethoxy)-phenyl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide,or a salt or polymorph thereof.

Embodiment 60: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)-phenyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 61: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 62: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 63: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 64: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 65: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 66: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)-pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 67: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)-pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 68: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 69: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 70: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises(R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)-pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof.

Embodiment 71: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide,or a salt or polymorph thereof.

Embodiment 72: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof.

Embodiment 73: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof.

Embodiment 74: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprisesN,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof.

Embodiment 75: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises Compound 1:

also known as2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3-(trifluoromethoxy)phenyl]acetamido}-pyridazin-3-yl)butyl]-1,3,4-thiadiazol-2-yl}acetamide(CB-839).

Embodiment 76: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises Compound 2:

also known as(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide.

Embodiment 77: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula I:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from C—H, C—F, and N;

R¹ and R⁴ are independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, C(R³)₂C(O)R³, C(R³)₂C(O)N(R³)₂, C(R³)₂N(R³)₂, C(R³)₂NR³C(O)R³,C(R³)₂NR³C(O)OR³, C(R³)₂NR³C(O)N(R³)₂, C(R³)₂NR³S(O)R³,C(R³)₂NR³S(O)₂R³, N(R³)₂, NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂,NR³S(O)R³, NR³S(O)₂R³, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)R³, SR³,S(O)R³, and S(O)₂R³, wherein each R¹ and R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

R² is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R¹ and R² together with the atoms to which theyare attached optionally form an form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁶)₂,NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂, NR⁶S(O)C(R⁶)₃,NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂, C(O)C(R⁶)₃,SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃;

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups; and

Z is heteroaryl, which may be optionally substituted.

Embodiment 78: The method of Embodiment 77, wherein:

Z is

Z¹ is chosen from C and N; and

Z², Z³, and Z⁴ are independently chosen from N, O, S, and CH, wherein atleast one of Z¹, Z², Z³, and Z⁴ is chosen from N, O, and S.

Embodiment 79: The method of Embodiment 77, wherein:

Z is

Z¹ is chosen from C and N;

Z² is chosen from N, CH, and C(O);

Z³, and Z⁴ are independently chosen from N and CH, wherein at least oneof Z¹, Z², Z³, and Z⁴ is N; and

R⁵ is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R⁴ and R⁵ together with the atoms to which theyare attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups.

Embodiment 80: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula II:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from N and CH;

A³ is chosen from N and CR²;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, C(O)N(R³)₂, and C(O)C(R³)₃,wherein R¹ may be optionally substituted with between 0 and 3 R^(z)groups;

R² is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(O)N(R³)₂, C(O)C(R³)₃, C(O)OH, C(O)OC(R³)₃, wherein R¹ and R² togetherwith the atoms to which they are attached optionally form an form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

R⁴ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,N(R³)₂, NR³C(O)C(R³)₃, NR³C(O)OC(R³)₃, NR³C(O)N(R³)₂, NR³S(O)C(R³)₃,NR³S(O)₂C(R³)₃, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)C(R³)₃,SC(R³)₃, S(O)C(R³)₃, and S(O)₂C(R³)₃, wherein R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

each R^(z) group is independently chosen from alkenyl, alkoxy, alkyl,aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, oxo, N(R⁶)₂, NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂,NR⁶S(O)C(R⁶)₃, NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂,C(O)C(R⁶)₃, SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃;

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups; and

Z is heteroaryl, which may be optionally substituted.

Embodiment 81: The method of Embodiment 80, wherein:

Z is

Z¹ is chosen from C and N; and

Z², Z³, and Z⁴ are independently chosen from N, O, S, and CH, wherein atleast one of Z¹, Z², Z³, and Z⁴ is chosen from N, O, and S.

Embodiment 82: The method of Embodiment 80, wherein:

Z is

Z¹ is chosen from C and N;

Z² is chosen from N, CH, and C(O);

Z³, and Z⁴ are independently chosen from N and CH, wherein at least oneof Z¹, Z², Z³, and Z⁴ is N; and

R⁵ is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R⁴ and R⁵ together with the atoms to which theyare attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups.

Embodiment 83: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula III:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(X) and R^(Y) is independently chosen from alkyl, cyano, H, andhalo, or two R^(X) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ is chosen from C and N;

A², A³, and A⁴ are independently chosen from N, O, S, and CH, wherein atleast one of A¹, A², A³, and A⁴ is chosen from N, O, and S;

R¹ and R² are each independently chosen from alkenyl, alkyl, aryl,arylalkyl, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹and R² each may be optionally substituted with one to three R^(Z)groups, wherein R¹ and R² together with the atoms to which they areattached optionally form an heteroaryl, or heterocycloalkyl ring, whichmay be optionally substituted with one to three R^(Z) groups;

R³ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(R⁴)₂C(O)R⁴, C(R⁴)₂C(O)N(R⁴)₂, C(R⁴)₂N(R⁴)₂, C(R⁴)₂NR⁴C(O)R⁴,C(R⁴)₂NR⁴C(O)OR⁴, C(R⁴)₂NR⁴C(O)N(R⁴)₂, C(R⁴)₂NR⁴S(O)R⁴,C(R⁴)₂NR⁴S(O)₂R⁴, N(R⁴)₂, NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂,NR⁴S(O)R⁴, NR⁴S(O)₂R⁴, C(O)N(R⁴)₂, S(O)N(R⁴)₂, S(O)₂N(R⁴)₂, C(O)R⁴, SR⁴,S(O)R⁴, and S(O)₂R⁴;

wherein each R³ may be optionally substituted with one to three R^(Z)groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R⁴ may be optionally substituted with one tothree R^(Z) groups, wherein two R⁴ groups together with the atoms towhich they are attached optionally form an heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with one tothree R^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵;

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups; and

Z is a monocyclic heteroaryl, which may be optionally substituted.

Embodiment 84: The method of Embodiment 83, wherein:

Z is

Z¹ is chosen from C and N; and

Z², Z³, and Z⁴ are independently chosen from N, O, S, and CH, wherein atleast one of Z¹, Z², Z³, and Z⁴ is chosen from N, O, and S.

Embodiment 85: The method of Embodiment 83, wherein:

Z is

Z¹ is C;

Z² is chosen from N and CH;

Z³, and Z⁴ are independently chosen from N and CH, wherein at least oneof Z¹, Z², Z³, and Z⁴ is N; and

R⁶ is chosen from, alkyl, cyano, cycloalkyl, H, halo, haloalkyl, andheterocycloalkyl, wherein R³ and R⁶ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted with oneto three R^(Z) groups.

Embodiment 86: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula IIIc:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

Embodiment 87: The method of Embodiment 86, wherein R¹ is methyl.

Embodiment 88: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula IIIc-1:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

Embodiment 89: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula IIIc-2:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

Embodiment 90: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula IIId:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

each of R^(Z1) and R^(Z2) is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, and oxo.

Embodiment 91: The method of Embodiment 90, wherein:

R^(X) is chosen from fluoro and H; and

each of R^(Z1) and R^(Z2) is independently chosen from alkyl,cycloalkyl, cycloalkylhaloalkyl, cycloalkyloxy, H, haloalkoxy,haloalkoxyaryl, haloalkyl, halocycloalkyloxy, heterocycloalkyl, andheterocycloalkyloxy.

Embodiment 92: The method of Embodiment 90, wherein:

R^(X) is chosen from fluoro and H; and

each of R^(Z1) and R^(Z2) is independently chosen from H,

Embodiment 93: The method of Embodiment 48, wherein said one or moreglutathione lowering agents comprises a compound of Formula IIIe:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R^(Z1) is chosen from alkenyl, alkoxy, alkoxyalkyl, alkoxyaryl,alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, and oxo.

Embodiment 94: The method of any one of Embodiments 48-50 and 52-93,wherein said one or more glutathione lowering agents comprises exactlyone GLS-1 inhibitor.

Provided below are exemplary embodiments of the disclosure.

Embodiment M-1: A method of treating cancer in a subject whose cancercells express low levels of asparagine synthetase (ASNS), as defined byan Histophathology Score (H-score) of less than or equal to 100 byimmunohistochemical staining, comprising administering a glutaminase-1(GLS-1) inhibitor to said subject.

Embodiment M-2: A method of treating a subject having a cancer or atumor in need of treatment comprising:

-   -   (a) determining the concentration or expression of ASNS in said        cancer or tumor of said subject; and    -   (b) administering a glutaminase-1 (GLS-1) inhibitor to said        subject if the level of ASNS is quantified as an H-score of less        than or equal to 100 by immunohistochemical staining.

Embodiment M-3: The method of Embodiment M-2, wherein the tumor iscancerous.

Embodiment M-4: The method of Embodiment M-1 or Embodiment M-3, whereinthe cancer is: bladder cancer, bone marrow cancer, breast cancer, cancerof the central nervous system, cervical cancer, colon cancer,endometrial cancer, cancer of the gastric system, head and neck cancer,kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, musclecancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,thyroid cancer, or a variant thereof.

Embodiment M-5: The method of Embodiment M-4, wherein the cancer isovarian, skin, liver, prostate, breast, colon, lung, head and neckcancers or a lymphoma.

Embodiment M-6: The method of Embodiment M-4, wherein the cancer isovarian cancer.

Embodiment M-7: The method of Embodiment M-6, wherein the ovarian canceris high-grade serous ovarian cancer (HGSOC).

Embodiment M-8: The method of Embodiment M-7, wherein the ovarian canceris nonresectable or relapsed HGSOC.

Embodiment M-9: The method of Embodiment M-1 or Embodiment M-2, whereinthe GLS-1 inhibitor is a selective inhibitor of GLS-1.

Embodiment M-10: The method of Embodiment M-1 or Embodiment M-2, whereinthe GLS-1 inhibitor binds an allosteric pocket on the solvent exposedregion of the GLS-1 dimer in the binding pocket present in the vicinityof Leu321, Phe322, Leu323, and Tyr394 from both monomers.

Embodiment M-11: The method of Embodiment M-1 or Embodiment M-2, whereinthe GLS-1 inhibitor is selected from the list of compounds provided inTable 1.

Embodiment M-12: The method of Embodiment M-1 or Embodiment M-2, whereinthe GLS-1 inhibitor is compound 1 or compound 2.

Embodiment M-13: The method of claim Embodiment M-1 or Embodiment M-2,wherein the subject is human.

Embodiment M-14: The method of claim Embodiment M-1 or Embodiment M-3,further comprising administering another pharmaceutically activecompound.

Embodiment M-15: The method of claim Embodiment M-14, wherein the otherpharmaceutically active compound is an anti-cancer agent.

Embodiment M-16: The method of claim Embodiment M-15, wherein theanti-cancer agent is chosen from a platinum-based agent, a taxane-basedagent, an immunotherapy, an immuno-oncotherapy, and a targeted therapy.

Embodiment M-17: The method of claim Embodiment M-15, wherein thetargeted therapy is an inhibitor of MEK kinase, HSP90, CDK4, or the mTORpathway.

Embodiment M-18: The method of claim Embodiment M-1 or Embodiment M-3,wherein the method further comprises administering non-chemical methodsof cancer treatment.

Embodiment M-19: The method of Embodiment M-18, wherein the methodfurther comprises administering radiation therapy.

Embodiment M-20: The method of Embodiment M-18, wherein the methodfurther comprises administering surgery, thermoablation, focusedultrasound therapy, cryotherapy, or any combination thereof.

Also provided herein is a GLS-1 inhibitor or a compound that inhibitsglutathione production for use as a medicament in the treatment of adisorder, for example, a tumor or cancer, in need of treatment, in asubject in whose tumors or cancer cells the concentration or expressionlevel of ASNS is low. In one embodiment, the tumor is cancerous and theconcentration or expression level of ASNS in the tumor of the subject islow.

The cancer may be any one of the types of cancers provided below. In oneembodiment, the cancer is ovarian, skin, liver, prostate, breast, colon,lung, head and neck cancers or a lymphoma. In another embodiment, thecancer is ovarian cancer, for example HGSOC, Epithelial, Germ CellTumor, and Low Malignant Potential Tumor.

Also provided herein is a GLS-1 inhibitor or a compound that inhibitsglutathione production for treatment of a disorder in need of treatment,in a subject in whose tumor or cancer cells the concentration orexpression level of ASNS is low.

Also provided herein is a GLS-1 inhibitor or a compound that inhibitsglutathione production for use in the manufacture of a medicament forthe treatment of a disorder in need of treatment, in a subject subjectin whose tumor or cancer cells the concentration or expression level ofASNS is low.

In certain embodiments, the disorder is a cancer. The cancer may be anycancer now known, or later discovered, including, but not limited to,Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma andLymphoma), Anal Cancer, Appendix Cancer, Atypical Teratoid/RhabdoidTumor, Basal Cell Carcinoma, Bile Duct Cancer (including Extrahepatic),Bladder Cancer, Bone Cancer (including Osteosarcoma and MalignantFibrous Histiocytoma), Brain Tumor (such as Astrocytomas, Brain andSpinal Cord Tumors, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors,Craniopharyngioma, Ependymoblastoma, Ependymoma, Medulloblastoma,Medulloepithelioma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, BasalCell Carcinoma, Bile Duct Cancer (including Extrahepatic), BladderCancer, Bone Cancer (including Osteosarcoma and Malignant FibrousHistiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary, CentralNervous System (such as Atypical Teratoid/Rhabdoid Tumor, EmbryonalTumors and Lymphoma), Cervical Cancer, Childhood Cancers, Chordoma,Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma (Mycosis Fungoides andSézary Syndrome), Duct, Bile (Extrahepatic), Ductal Carcinoma In Situ(DCIS), Embryonal Tumors (Central Nervous System), Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer(like Intraocular Melanoma, Retinoblastoma), Fibrous Histiocytoma ofBone (including Malignant and Osteosarcoma), Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalStromal Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia,Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors (Endocrine, Pancreas), KaposiSarcoma, Kidney (including Renal Cell), Langerhans Cell Histiocytosis,Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), AcuteMyeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML),Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSézary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer (such as Epithelial, GermCell Tumor, Low Malignant Potential Tumor and High Grade Serous OvarianCancer), Pancreatic Cancer (including Islet Cell Tumors),Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma,Pineal Parenchymal Tumors of Intermediate Differentiation, Pineoblastomaand Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor,Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma,Pregnancy and Breast Cancer, Primary Central Nervous System (CNS)Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer,Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma,Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (like Ewing SarcomaFamily of Tumors, Kaposi, Soft Tissue, Uterine), Sézary Syndrome, SkinCancer (such as Melanoma, Merkel Cell Carcinoma, Nonmelanoma), SmallCell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, SquamousCell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic,Stomach (Gastric) Cancer, Supratentorial Primitive NeuroectodermalTumors, T-Cell Lymphoma (Cutaneous, Mycosis Fungoides and SézarySyndrome), Testicular Cancer, Throat Cancer, Thymoma and ThymicCarcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvisand Ureter, Trophoblastic Tumor (Gestational), Unknown Primary, UnusualCancers of Childhood, Ureter and Renal Pelvis, Transitional Cell Cancer,Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma,Waldenström Macroglobulinemia and Wilms Tumor, or a variant thereof.

In certain embodiments, the cancer is ovarian, skin, liver, prostate,breast, colon, lung, head and neck cancers, a lymphoma, or a variantthereof.

In certain embodiments, the cancer is ovarian cancer.

In certain embodiments, the cancer is high-grade serous ovarian cancer(HGSOC).

In certain embodiments, the cancer is nonresectable or relapsed HGSOC.

In some embodiments of the invention, the invention comprises a methodof treating a tumor or a cancer in a subject whose tumor or cancer cellsexpress low levels of ASNS comprising administering a glutaminaseinhibitor to said subject. In one embodiment, the level of ASNS asmeasured by immunohistochemistry is quantified with an H-score of lessthan or equal to 100. The cancer may be any of the cancers listed above.In one embodiment, the cancer is ovarian, skin, liver, prostate, breast,colon, lung, head and neck cancers or a lymphoma. In another embodiment,the cancer is ovarian cancer, for example, high-grade serous ovariancancer (HGSOC). In yet another embodiment, the cancer is nonresectableor relapsed HGSOC.

As provided above, the inventors have discovered that cells, forexample, cancer or tumor cells, that express high levels of ASNS areresistant to inhibition of GLS-1. Inhibition of GLS-1 reduces the steadystate levels of glutathione, thus shifting the redox balance of thecells. This inhibits cell growth through a mechanism that involvesreactive oxygen species (ROS)-induced DNA damage. Asparagine synthetaseor ASNS or aspartate-ammonia ligase is a chiefly cytoplasmic enzyme thatgenerates asparagine from aspartate, while converting glutamine toglutamate. Attempts to reduce cellular levels of glutamate (andultimately glutathione) by GLS-1 inhibitors would have little, or no,overall effect if the reduction is countered through the activity ofASNS, and ASNS-derived glutamate to produce glutathione.

In order for cells, such as cancer or tumor cells, to respond totreatment with a GLS-1 inhibitor (GLS1i), the levels of ASNS must bebelow certain levels. In one embodiment, the level of ASNS is quantifiedby immunohistochemistry and calculated as an H-score. An H-score of0-100 (inclusive) or less than or equal to 100 would indicate responseto GLS inhibition.

Calculation of a Histophathology Score or H-score is known to one ofskill in the art. In one embodiment, expression of ASNS protein in tumorcells is detected or measured, for example, using microscopy andimmunohistochemistry (IHC) and the H-score is determined therefrom.Based on the intensity of staining, the sample is, for example, scoredat 4 different levels, on a scale of 0 to 3+, for ASNS proteinexpression.

As used herein the term “H-score” is used to mean an immunohistologyscore for ASNS expression in a tumor sample. In an attempt to accuratelydescribe the extent of immunohistochemical staining of a tumor, thedegree of IHC staining, if any, in each sub-cellular compartment intumor cells is captured for ASNS. As further described below, the degreeor intensity of staining is classified into 4 levels that are assigned ascore from 0 (no staining) to 3+ (greatest degree or most intensestaining).

This algorithm includes capturing the percentage of tumor cells stainedat each intensity level. A semi-quantitative intensity scale rangingfrom 0 for no staining, to 3+ for the most intense staining, is used.All of this information is used to calculate the H-Score. This score ismore representative of the staining of the entire tumor on the section.Although given sections may share the same simple intensity score, thereis a difference between a 3+ case with only 10% of the cells staining ascompared to a 3+ case where greater than 90% of the cells are staining.This difference is easily picked up using the H-Score method. An H-Scoreis typically calculated for staining of each sub-cellular compartmentfor both normal and tumor cells using the following formula; H-Score=(%cells at 0)*0+ (% cells at 1+)*1+ (% cells at 2+)*2+. (% cells at 3+)*3.Thus, this score produces a continuous variable that ranges from 0 to300. An H-score of 0-100 would be considered 1+, an H-score of 101-200would be scored 2+, and an H-score of 201-300 scored 3+.

In some embodiments, the tumor or cancer express low levels of ASNS, asdetermined by the H-score. In one embodiment, the H-score is 0-150 (bothinclusive). In another embodiment, the H-score is less than or equal to150. In another embodiment, the H-score is 0-125 (both inclusive). Inanother embodiment, the H-score is less than or equal to 125. In someembodiments, the H-score is 0-100 (both inclusive). In some embodiments,the H-score is less than or equal to 100. In some embodiments, theH-score is less than 100.

In some embodiments, the H-score is less than or equal to 95. In someembodiments, the H-score is less than or equal to 90. In someembodiments, the H-score is less than or equal to 85. In someembodiments, the H-score is less than or equal to 80. In someembodiments, the H-score is less than or equal to 75. In someembodiments, the H scose is less than or equal to 70. In someembodiments, the H-score is less than or equal to 65. In someembodiments, the H-score is less than or equal to 60. In someembodiments, the H-score is less than or equal to 55. In someembodiments, the H-score is less than or equal to 50. In someembodiments, the H-score is less than or equal to 45. In someembodiments, the H-score is less than or equal to 40. In someembodiments, the H-score is less than or equal to 35. In someembodiments, the H-score is less than or equal to 30. In someembodiments, the H-score is less than or equal to 25. In someembodiments, the H-score is less than or equal to 20. In someembodiments, the H-score is less than or equal to 15. In someembodiments, the H-score is less than or equal to 10.

In certain embodiments, the GLS1i is a selective inhibitor of GLS-1.

In certain embodiments, the GLS1i binds an allosteric pocket on thesolvent exposed region of the GLS-1 dimer in the binding pocket presentin the vicinity of Leu321, Phe322, Leu323, and Tyr394 from bothmonomers.

In certain embodiments, the GLS1i is compound 1. In certain embodiments,the GLS1i is compound 2. In certain embodiments, the GLS-1 inhibitor isselected from the list of compounds provided in Table 1 below. Incertain embodiments, the compound is chosen from any combination of thecompounds provided in Table 1, or a salt or polymorph thereof. Forexample, in certain embodiments, the GLS1i is chosen from any two,three, four, five, six, seven, eight, none or ten of the compoundsprovided in Table 1, or a salt or polymorph thereof.

In certain embodiments, the subject is human.

In certain embodiments, methods disclosed herein further compriseadministering another pharmaceutically active compound. In certainembodiments, the disorder to be treated is a cancer and the otherpharmaceutically active compound is an anti-cancer agent. In certainembodiments, the anti-cancer agent is a chosen from a platinum-basedagent, a taxane-based agent, an immunotherapy, an immune-oa targetedtherapy. In certain embodiments, the targeted therapy is an inhibitor ofMEK kinase, HSP90, CDK4, or the mTOR pathway.

In certain embodiments, methods disclosed herein further compriseadministering non-chemical methods of cancer treatment. In certainembodiments, the method further comprises administering radiationtherapy. In certain embodiments, the method further comprisesadministering surgery, thermoablation, focused ultrasound therapy,cryotherapy, or any combination thereof.

In certain embodiments, methods disclosed herein administer the activeagent (e.g., a compound that inhibits glutathione production,glutaminase-1 inhibitor, or selective GLS1i) as a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient orcarrier. In certain embodiments, the pharmaceutical composition isformulated for oral administration. In certain embodiments,pharmaceutical composition is formulated as a tablet or capsule. Incertain embodiments, the pharmaceutical composition is formulated forparenteral administration.

Also provided are embodiments wherein any embodiment disclosed herein,may be combined with any one or more of these embodiments to form a newcompound or class of compounds, or pharmaceutical composition comprisingit, or method of use employing it, provided the combination is notmutually exclusive. For example, a combination embodiment wherein thesubject is human and the disorder in need of treatment is cancer isvalid because the recited limitations are not mutually exclusive.

Abbreviations and Definitions

To facilitate understanding of the disclosure, a number of terms andabbreviations as used herein are defined below as follows:

When introducing elements of the present disclosure or the preferredembodiment(s) thereof, the articles “a,” “an,” “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B, i.e., A alone,B alone or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination or A, B, and C incombination.

When ranges of values are disclosed, and the notation “from n1 . . . ton2” or “between n1 . . . and n2” is used, where n1 and n2 are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about,” as used herein in relation to a numerical value xmeans x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The terms “GLS-1 inhibitor” and “GLS1i” are used interchangeably hereinto refer to a compound that exhibits an IC₅₀ with respect to GLS-1activity of no more than about 100 μM and more typically not more thanabout 50 μM, as measured in the GLS-1 enzyme assay described generallyherein below. IC₅₀ is that concentration of inhibitor that reduces theactivity of an enzyme (e.g., GLS-1) to half-maximal level. Certaincompounds disclosed herein have been discovered to exhibit inhibitionagainst GLS-1. In certain embodiments, compounds will exhibit an IC₅₀with respect to GLS-1 of no more than about 10 μM; in furtherembodiments, compounds will exhibit an IC₅₀ with respect to GLS-1 of nomore than about 5 μM; in yet further embodiments, compounds will exhibitan IC₅₀ with respect to GLS-1 of not more than about 1 μM; in yetfurther embodiments, compounds will exhibit an IC₅₀ with respect toGLS-1 of not more than about 200 nM, as measured in the GLS-1 enzymaticassay described herein.

The terms “inhibitor selective for GLS-1” and a “selective inhibitor ofGLS-1” are used interchangeably herein and refer to inhibitors that areabout 100 times more selective for GLS-1 than for GLS-2 as measured inany assay known to one of skill in the art that measures the activity ofthe enzyme. An example of such an assay includes, but is not limited to,the GLS-1 enzyme assay (GLS-1 Enzymatic Activity Assay) described below.

The term “glutathione lowering agent” is used herein to refer to acompound that reduces glutathione levels. In certain embodiments, theglutathione lowering agent inhibits amino acid or glutathione transport.In certain embodiments, the glutathione lowering agent inhibits aminoacid or glutathione activity. In certain embodiments, the compound is aglutaminase inhibitor. In certain embodiments, the glutathione loweringagent is a GLS-1 inhibitor.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prevention, prophylaxis, attenuation, amelioration and therapy.Treatment may also include prevention of disease. Prevention of adisease may involve complete protection from disease, for example as inthe case of prevention of infection with a pathogen, or may involveprevention of disease progression. For example, prevention of a diseasemay not mean complete foreclosure of any effect related to the diseasesat any level, but instead may mean prevention of the symptoms of adisease to a clinically significant or detectable level. Prevention ofdiseases may also mean prevention of progression of a disease to a laterstage of the disease.

The terms “subject” and “patient” are used interchangeably herein tomean all mammals including humans. Examples of subjects include, but arenot limited to, humans, monkeys, dogs, cats, horses, cows, goats, sheep,pigs, and rabbits. In one embodiment, the patient is a human.

The terms “affected with a disease or disorder,” “afflicted with adisease or disorder,” and “having a disease or disorder” are usedinterchangeably herein and refer to a subject or patient with anydisease, disorder, syndrome or condition. No increased or decreasedlevel of severity of the disorder is implied by the use of one the termsas compared to the other.

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycle, or any other moiety were the atom attached to the carbonylis carbon. An “acetyl” group refers to a —C(O)CH₃ group. An“alkylcarbonyl” or “alkanoyl” group refers to an alkyl group attached tothe parent molecular moiety through a carbonyl group. Examples of suchgroups include methylcarbonyl and ethylcarbonyl. Examples of acyl groupsinclude formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, the alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—), (—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, the alkyl will comprise from 1 to10 carbon atoms. In further embodiments, the alkyl will comprise from 1to 6 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, noyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH₂—). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R—S—) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, the alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, the alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:::C—, —C═C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(O)N(RR′) group with R and R′ as defined herein or as defined by thespecifically enumerated “R” groups designated. The term “N-amido” asused herein, alone or in combination, refers to a RC(O)N(R′)— group,with R and R′ as defined herein or as defined by the specificallyenumerated “R” groups designated. The term “acylamino” as used herein,alone or in combination, embraces an acyl group attached to the parentmoiety through an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted. Additionally, R and R′ may combine to formheterocycloalkyl, either of which may be optionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch polycyclic ring systems are fused together. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl.

The term “arylalkenyl” or “aralkenyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkenyl group.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkynyl” or “aralkynyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl,phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term “aryloxy” as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C6H4=derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′— group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxy,” as used herein, refers to —C(O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt. An “O-carboxy” group refers to a RC(O)O— group, where R is asdefined herein. A “C-carboxy” group refers to a —C(O)OR groups where Ris as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl group wherein each cyclic moietycontains from 3 to 12 carbon atom ring members and which may optionallybe a benzo fused ring system which is optionally substituted as definedherein. In certain embodiments, the cycloalkyl will comprise from 5 to 7carbon atoms. Examples of such cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronapthyl,indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and thelike. “Bicyclic” and “tricyclic” as used herein are intended to includeboth fused ring systems, such as decahydronaphthalene,octahydronaphthalene as well as the multicyclic (multicentered)saturated or partially unsaturated type. The latter type of isomer isexemplified in general by, bicyclo[1,1,1]pentane, camphor, adamantane,and bicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF2-), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto a 3 to 15 membered unsaturated heteromonocyclic ring, or a fusedmonocyclic, bicyclic, or tricyclic ring system in which at least one ofthe fused rings is aromatic, which contains at least one atom selectedfrom the group consisting of O, S, and N. In certain embodiments, theheteroaryl will comprise from 5 to 7 carbon atoms. The term alsoembraces fused polycyclic groups wherein heterocyclic rings are fusedwith aryl rings, wherein heteroaryl rings are fused with otherheteroaryl rings, wherein heteroaryl rings are fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic group containing at least one heteroatom as aring member, wherein each the heteroatom may be independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur In certainembodiments, the hetercycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, the hetercycloalkyl willcomprise from 1 to 2 heteroatoms as ring members. In certainembodiments, the hetercycloalkyl will comprise from 3 to 8 ring membersin each ring. In further embodiments, the hetercycloalkyl will comprisefrom 3 to 7 ring members in each ring. In yet further embodiments, thehetercycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Examples ofheterocycle groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl,dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl,dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl,benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl,1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl,pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and thelike. The heterocycle groups may be optionally substituted unlessspecifically prohibited.

The term “hydrazinyl” as used herein, alone or in combination, refers totwo amino groups joined by a single bond, i.e., —N—N—.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The phrase “in the main chain” refers to the longest contiguous oradjacent chain of carbon atoms starting at the point of attachment of agroup to the compounds of any one of the formulas disclosed herein.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms.

The term “lower aryl,” as used herein, alone or in combination, meansphenyl or naphthyl, either of which may be optionally substituted asprovided.

The term “lower heteroaryl,” as used herein, alone or in combination,means either 1) monocyclic heteroaryl comprising five or six ringmembers, of which between one and four the members may be heteroatomsselected from the group consisting of O, S, and N, or 2) bicyclicheteroaryl, wherein each of the fused rings comprises five or six ringmembers, comprising between them one to four heteroatoms selected fromthe group consisting of O, S, and N.

The term “lower cycloalkyl,” as used herein, alone or in combination,means a monocyclic cycloalkyl having between three and six ring members.Lower cycloalkyls may be unsaturated. Examples of lower cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “lower heterocycloalkyl,” as used herein, alone or incombination, means a monocyclic heterocycloalkyl having between threeand six ring members, of which between one and four may be heteroatomsselected from the group consisting of O, S, and N. Examples of lowerheterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl,piperidinyl, piperazinyl, and morpholinyl. Lower heterocycloalkyls maybe unsaturated.

The term “lower amino,” as used herein, alone or in combination, refersto —NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, lower alkyl, and lower heteroalkyl, any of whichmay be optionally substituted. Additionally, the R and R′ of a loweramino group may combine to form a five- or six-memberedheterocycloalkyl, either of which may be optionally substituted.

The term “mercaptyl” as used herein, alone or in combination, refers toan RS-group, where R is as defined herein.

The term “nitro,” as used herein, alone or in combination, refers to—NO2.

The terms “oxy” or “oxa,” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refer the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocarbamyr refers to a —OC(S)NRR”, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

The term “trisubstituted silyl,” as used herein, alone or incombination, refers to a silicone group substituted at its three freevalences with groups as listed herein under the definition ofsubstituted amino. Examples include trimethysilyl,tert-butyldimethylsilyl, triphenylsilyl and the like.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that the group isabsent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five-, six-, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which may be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R, R′ and Rn where n=(1, 2,3, . . . n), every substituent, and every term should be understood tobe independent of every other in terms of selection from a group. Shouldany variable, substituent, or term (e.g. aryl, heterocycle, R, etc.)occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups may be attached to a parent molecule or may occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— may beattached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the disclosure encompasses all stereochemicalisomeric forms, including diastereomeric, enantiomeric, and epimericforms, as well as d-isomers and 1-isomers, and mixtures thereof.Individual stereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentdisclosure includes all cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers as well as the appropriate mixtures thereof.Additionally, compounds may exist as tautomers; all tautomeric isomersare provided by this disclosure. Additionally, the compounds disclosedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. In general, the solvated forms are considered equivalent to theunsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered part of largersubstructure. A bond may be single, double, or triple unless otherwisespecified. A dashed line between two atoms in a drawing of a moleculeindicates that an additional bond may be present or absent at thatposition.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present disclosure includes compounds listed above in theform of salts, including acid addition salts. Suitable salts includethose formed with both organic and inorganic acids. Such acid additionsalts will normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent disclosure contemplates sodium, potassium, magnesium, andcalcium salts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid.

Compounds

Examples of compounds useful in the methods of the invention areprovided herein. In one embodiment, the compound is a compound thatinhibits glutathione production or activity. In certain embodiments, thecompound inhibits amino acid or glutathione transport. In someembodiments, the compound is a glutaminase inhibitor.

In certain embodiments, the compound is a GLS-1 inhibitor, for example,a selective inhibitor of GLS-1. It is known that GLS-1 forms a tetramer(PNAS 2012, 109, 7705). In certain embodiments, the GLS1i occupies anallosteric pocket on the solvent exposed region between two GLS-1dimers. The GLS1i may, for example, bind GLS-1 in an allosteric pocketon the solvent exposed region of the GLS-1 dimer in the binding pocketpresent in the vicinity of amino acids Leu 321, Phe322, Leu323, andTyr394 from both monomers. Without being bound by any theory, theinventors propose that key interactions are made within a hydrophobiccluster that comprises Leu321, Phe322, Leu323, and Tyr394 from bothmonomers which forms the allosteric pocket. Binding of the glutaminaseinhibitor, for example, a GLS1i, induces a dramatic conformationalchange near the catalytic site rendering the enzyme inactive.

Compounds which inhibit GLS-1 are known in the art and disclosed herein.In certain embodiments, the compound is compound 1,

also known as2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3-(trifluoromethoxy)phenyl]acetamido}pyridazin-3-yl)butyl]-1,3,4-thiadiazol-2-yl}acetamide(CB-839).

In certain embodiments, the compound is compound 2,

also known as(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)-6-methylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide.

In certain embodiments, the compound (and its molecular mass) isprovided in Table 1 below, or a salt or polymorph thereof. In certainembodiments, the compound is chosen from any combination of thecompounds provided in Table 1 below, or a salt or polymorph thereof. Forexample, in certain embodiments, the compound is chosen from any two,three, four, five, six, seven, eight, nine or ten of the compoundsprovided in Table 1 below, or a salt or polymorph thereof.

TABLE 1 Compound MS (S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3- 586(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamideN,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2- 524diyl))bis(2-phenylacetamide), also known as BPTES2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3- 587(trifluoromethoxy)phenyl]acetamido}pyridazin-3-yl)butyl}1,3,4-thiadiazol-2-yl}acetamide, also known as CB-839N,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-558 diyl))bis(2-(pyridin-2-yl)acetamide)N-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)phenyl]pyridin-2- 554yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-563((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-480 yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamideN-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3 - 584(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)pyrrolidin-l-yl)-1,3,4-thiadiazole-2-carboxamide(R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)phenyl)pyridin-2- 572yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide (R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2- 480yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2- 494yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide (R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2- 502yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide (R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2- 518yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-561 yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-464 methyl-1H-1,2,3-triazole-4-carboxamide1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)-2- 482fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-500 yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-518 yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide(R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-452 fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-l-yl)-N-((4-569(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamideN,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-518 phenylacetamide)(both or either of 1S,3S and 1R,2R enantiomers)

In certain embodiments, the compound is(S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,or a salt or polymorph thereof. In certain embodiments, the compound isN,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof. In certain embodiments, the compound is(S)-2-hydroxy-2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,or a salt or polymorph thereof. In certain embodiments, the compound isN,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide),or a salt or polymorph thereof. In certain embodiments, the compound isN-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)phenyl]pyridin-2-yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound isN-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)phenyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is(R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound is5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide,or a salt or polymorph thereof. In certain embodiments, the compound isN,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof. In certain embodiments, the compound isN,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof. In certain embodiments, the compound isN,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),or a salt or polymorph thereof.

Additional non-limiting examples of compounds useful in the methods ofthe invention include the following compounds and pharmaceuticallyacceptable salts thereof.

In certain embodiments, the GLS1i is disclosed in United States PatentApplication Publication No. US2016/0002204 published Jan. 7, 2016.

In certain embodiments, the GLS-1 inhibitor is of Formula I:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from C—H, C—F, and N;

R¹ and R⁴ are independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, C(R³)₂C(O)R³, C(R³)₂C(O)N(R³)₂, C(R³)₂N(R³)₂, C(R³)₂NR³C(O)R³,C(R³)₂NR³C(O)OR³, C(R³)₂NR³C(O)N(R³)₂, C(R³)₂NR³S(O)R³,C(R³)₂NR³S(O)₂R³, N(R³)₂, NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂,NR³S(O)R³, NR³S(O)₂R³, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)R³, SR³,S(O)R³, and S(O)₂R³, wherein each R¹ and R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

R² is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R¹ and R² together with the atoms to which theyare attached optionally form an form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁶)₂,NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂, NR⁶S(O)C(R⁶)₃,NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂, C(O)C(R⁶)₃,SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃;

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups; and

Z is heteroaryl, which may be optionally substituted.

In certain embodiments, the GLS-1 inhibitor is of Formula Ia:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R¹ groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from C—H, C—F, and N;

Z¹ is chosen from C and N;

Z², Z³, and Z⁴ are independently chosen from N, O, S, and CH, wherein atleast one of Z¹, Z², Z³, and Z⁴ is chosen from N, O, and S;

R¹ and R⁴ are independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, C(R³)₂C(O)R³, C(R³)₂C(O)N(R³)₂, C(R³)₂N(R³)₂, C(R³)₂NR³C(O)R³,C(R³)₂NR³C(O)OR³, C(R³)₂NR³C(O)N(R³)₂, C(R³)₂NR³S(O)R³,C(R³)₂NR³S(O)₂R³, N(R³)₂, NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂,NR³S(O)R³, NR³S(O)₂R³, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)R³, SR³,S(O)R³, and S(O)₂R³, wherein each R¹ and R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

R² is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R¹ and R² together with the atoms to which theyare attached optionally form an form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁶)₂,NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂, NR⁶S(O)C(R⁶)₃,NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂, C(O)C(R⁶)₃,SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; and

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups.

In certain embodiments, the GLS-1 inhibitor is of Formula Ib:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from C—H, C—F, and N;

Z¹ is chosen from C and N;

Z² is chosen from N, CH, and C(O);

Z³, and Z⁴ are independently chosen from N and CH, wherein at least oneof Z¹, Z², Z³, and Z⁴ is N;

R¹ and R⁴ are independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, C(R³)₂C(O)R³, C(R³)₂C(O)N(R³)₂, C(R³)₂N(R³)₂, C(R³)₂NR³C(O)R³,C(R³)₂NR³C(O)OR³, C(R³)₂NR³C(O)N(R³)₂, C(R³)₂NR³S(O)R³,C(R³)₂NR³S(O)₂R³, N(R³)₂, NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂,NR³S(O)R³, NR³S(O)₂R³, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)R³, SR³,S(O)R³, and S(O)₂R³, wherein each R¹ and R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

R² and R⁵ are chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H,halo, and haloalkyl, wherein R¹ and R² together with the atoms to whichthey are attached optionally form an form an aryl, cycloalkyl,heteroaryl, or heterocycloalkyl ring, which may be optionallysubstituted with between 0 and 3 R^(z) groups, wherein R⁴ and R⁵together with the atoms to which they are attached optionally form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁶)₂,NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂, NR⁶S(O)C(R⁶)₃,NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂, C(O)C(R⁶)₃,SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; and

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups.

In certain embodiments, the GLS1i is disclosed in United States PatentApplication Publication No. US 2016/0002248, published Jan. 7, 2016.

In certain embodiments, the GLS-1 inhibitor is of Formula II:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from N and CH;

A³ is chosen from N and CR²;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, C(O)N(R³)₂, and C(O)C(R³)₃,wherein R¹ may be optionally substituted with between 0 and 3 R^(z)groups;

R² is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(O)N(R³)₂, C(O)C(R³)₃, C(O)OH, C(O)OC(R³)₃, wherein R¹ and R² togetherwith the atoms to which they are attached optionally form an form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

R⁴ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,N(R³)₂, NR³C(O)C(R³)₃, NR³C(O)OC(R³)₃, NR³C(O)N(R³)₂, NR³S(O)C(R³)₃,NR³S(O)₂C(R³)₃, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)C(R³)₃,SC(R³)₃, S(O)C(R³)₃, and S(O)₂C(R³)₃, wherein R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

each R^(z) group is independently chosen from alkenyl, alkoxy, alkyl,aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, oxo, N(R⁶)₂, NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂,NR⁶S(O)C(R⁶)₃, NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂,C(O)C(R⁶)₃, SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃;

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups; and

Z is heteroaryl, which may be optionally substituted.

In certain embodiments, the GLS-1 inhibitor is of Formula IIa:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from N and CH;

A³ is chosen from N and CR²;

Z¹ is chosen from C and N;

Z², Z³, and Z⁴ are independently chosen from N, O, S, and CH, wherein atleast one of Z¹, Z², Z³, and Z⁴ is chosen from N, O, and S;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, C(O)N(R³)₂, and C(O)C(R³)₃,wherein R¹ may be optionally substituted with between 0 and 3 R^(z)groups;

R² is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(O)N(R³)₂, C(O)C(R³)₃, C(O)OH, C(O)OC(R³)₃, wherein R¹ and R² togetherwith the atoms to which they are attached optionally form an form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

R⁴ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,N(R³)₂, NR³C(O)C(R³)₃, NR³C(O)OC(R³)₃, NR³C(O)N(R³)₂, NR³S(O)C(R³)₃,NR³S(O)₂C(R³)₃, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)C(R³)₃,SC(R³)₃, S(O)C(R³)₃, and S(O)₂C(R³)₃, wherein R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

each R^(z) group is independently chosen from alkenyl, alkoxy, alkyl,aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, oxo, N(R⁶)₂, NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂,NR⁶S(O)C(R⁶)₃, NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂,C(O)C(R⁶)₃, SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; and

each R⁶ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein two R⁶ groups together with the atoms to whichthey are attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(x) groups.

In certain embodiments, the GLS-1 inhibitor is of Formula IIb:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(x) and R^(y) is independently chosen from alkyl, cyano, H, andhalo, wherein two R^(x) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and A² are independently chosen from N and CH;

A³ is chosen from N and CR²;

Z¹ is chosen from C and N;

Z² is chosen from N, CH, and C(O);

Z³, and Z⁴ are independently chosen from N and CH, wherein at least oneof Z¹, Z², Z³, and Z⁴ is N;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, C(O)N(R³)₂, and C(O)C(R³)₃,wherein R¹ may be optionally substituted with between 0 and 3 R^(z)groups;

R² is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(O)N(R³)₂, C(O)C(R³)₃, C(O)OH, C(O)OC(R³)₃, wherein R¹ and R² togetherwith the atoms to which they are attached optionally form an form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups;

each R³ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R³ may be optionally substituted with between0 and 3 R^(z) groups, wherein two R³ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted withbetween 0 and 3 R^(z) groups;

R⁴ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,N(R³)₂, NR³C(O)C(R³)₃, NR³C(O)OC(R³)₃, NR³C(O)N(R³)₂, NR³S(O)C(R³)₃,NR³S(O)₂C(R³)₃, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂, C(O)C(R³)₃,SC(R³)₃, S(O)C(R³)₃, and S(O)₂C(R³)₃, wherein R⁴ may be optionallysubstituted with between 0 and 3 R^(z) groups;

R⁵ is chosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo,and haloalkyl, wherein R⁴ and R⁵ together with the atoms to which theyare attached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups;

each R^(z) group is independently chosen from alkenyl, alkoxy, alkyl,aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, oxo, N(R⁶)₂, NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂,NR⁶S(O)C(R⁶)₃, NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂,C(O)C(R⁶)₃, SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; and each R⁶ isindependently chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl,cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, and hydroxyl,wherein two R⁶ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with between 0 and 3 R^(x)groups.

In certain embodiments, the GLS1i is disclosed in United States PatentApplication Publication No. US 2016/0009704, published Jan. 14, 2016.

In certain embodiments, the GLS-1 inhibitor is of Formula III:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(X) and R^(Y) is independently chosen from alkyl, cyano, H, andhalo, or two R^(X) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ is chosen from C and N;

A², A³, and A⁴ are independently chosen from N, O, S, and CH, wherein atleast one of A¹, A², A³, and A⁴ is chosen from N, O, and S;

R¹ and R² are each independently chosen from alkenyl, alkyl, aryl,arylalkyl, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹and R² each may be optionally substituted with one to three R^(Z)groups, wherein R¹ and R² together with the atoms to which they areattached optionally form an heteroaryl, or heterocycloalkyl ring, whichmay be optionally substituted with one to three R^(Z) groups;

R³ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(R⁴)₂C(O)R⁴, C(R⁴)₂C(O)N(R⁴)₂, C(R⁴)₂N(R⁴)₂, C(R⁴)₂NR⁴C(O)R⁴,C(R⁴)₂NR⁴C(O)OR⁴, C(R⁴)₂NR⁴C(O)N(R⁴)₂, C(R⁴)₂NR⁴S(O)R⁴,C(R⁴)₂NR⁴S(O)₂R⁴, N(R⁴)₂, NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂,NR⁴S(O)R⁴, NR⁴S(O)₂R⁴, C(O)N(R⁴)₂, S(O)N(R⁴)₂, S(O)₂N(R⁴)₂, C(O)R⁴, SR⁴,S(O)R⁴, and S(O)₂R⁴;

wherein each R³ may be optionally substituted with one to three R^(Z)groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R⁴ may be optionally substituted with one tothree R^(Z) groups, wherein two R⁴ groups together with the atoms towhich they are attached optionally form an heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with one tothree R^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵;

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups; and

Z is a monocyclic heteroaryl, which may be optionally substituted.

In certain embodiments, the GLS-1 inhibitor is of Formula IIIa:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(X) and R^(Y) is independently chosen from alkyl, cyano, H, andhalo, or two R^(X) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ and Z¹ are independently chosen from C and N;

A², A³, A⁴, Z², Z³, and Z⁴ are independently chosen from N, O, S, andCH, wherein at least one of A¹, A², A³, and A⁴ and at least one of Z¹,Z², Z³, and Z⁴ is chosen from N, O, and S;

R¹ and R² are each independently chosen from alkenyl, alkyl, aryl,arylalkyl, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹and R² each may be optionally substituted with one to three R^(Z)groups, wherein R¹ and R² together with the atoms to which they areattached optionally form an heteroaryl, or heterocycloalkyl ring, whichmay be optionally substituted with one to three R^(Z) groups;

R³ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(R⁴)₂C(O)R⁴, C(R⁴)₂C(O)N(R⁴)₂, C(R⁴)₂N(R⁴)₂, C(R⁴)₂NR⁴C(O)R⁴,C(R⁴)₂NR⁴C(O)OR⁴, C(R⁴)₂NR⁴C(O)N(R⁴)₂, C(R⁴)₂NR⁴S(O)R⁴,C(R⁴)₂NR⁴S(O)₂R⁴, N(R⁴)₂, NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂,NR⁴S(O)R⁴, NR⁴S(O)₂R⁴, C(O)N(R⁴)₂, S(O)N(R⁴)₂, S(O)₂N(R⁴)₂, C(O)R⁴, SR⁴,S(O)R⁴, and S(O)₂R⁴;

wherein each R³ may be optionally substituted with one to three R^(Z)groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R⁴ may be optionally substituted with one tothree R^(Z) groups, wherein two R⁴ groups together with the atoms towhich they are attached optionally form an heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with one tothree R^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵;

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

In certain embodiments, the GLS-1 inhibitor is of Formula IIIb:

or a salt thereof, wherein:

n is chosen from 3, 4, and 5;

each R^(X) and R^(Y) is independently chosen from alkyl, cyano, H, andhalo, or two R^(X) groups together with the atoms to which they areattached optionally form a cycloalkyl ring;

A¹ is chosen from C and N;

A², A³, and A⁴, are independently chosen from N, O, S, and CH, whereinat least one of A¹, A², A³, and A⁴ is chosen from N, O, and S;

Z¹ is C;

Z², Z³ and Z⁴ are independently chosen from N and CH, wherein at leastone of Z¹, Z², Z³, and Z⁴ is N;

R¹ and R² are each independently chosen from alkenyl, alkyl, aryl,arylalkyl, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹and R² each may be optionally substituted with one to three R^(Z)groups, wherein R¹ and R² together with the atoms to which they areattached optionally form an heteroaryl, or heterocycloalkyl ring, whichmay be optionally substituted with one to three R^(Z) groups;

R³ is chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano,cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, hydroxyl,C(R⁴)₂C(O)R⁴, C(R⁴)₂C(O)N(R⁴)₂, C(R⁴)₂N(R⁴)₂, C(R⁴)₂NR⁴C(O)R⁴,C(R⁴)₂NR⁴C(O)OR⁴, C(R⁴)₂NR⁴C(O)N(R⁴)₂, C(R⁴)₂NR⁴S(O)R⁴,C(R⁴)₂NR⁴S(O)₂R⁴, N(R⁴)₂, NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂,NR⁴S(O)R⁴, NR⁴S(O)₂R⁴, C(O)N(R⁴)₂, S(O)N(R⁴)₂, S(O)₂N(R⁴)₂, C(O)R⁴, SR⁴,S(O)R⁴, and S(O)₂R⁴;

wherein each R³ may be optionally substituted with one to three R^(Z)groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein each R⁴ may be optionally substituted with one tothree R^(Z) groups, wherein two R⁴ groups together with the atoms towhich they are attached optionally form an heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with one tothree R^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵;

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups; and

R⁶ is chosen from, alkyl, cyano, cycloalkyl, H, halo, haloalkyl, andheterocycloalkyl, wherein R³ and R⁶ groups together with the atoms towhich they are attached optionally form an aryl, cycloalkyl, heteroaryl,or heterocycloalkyl ring, which may be optionally substituted with oneto three R^(Z) groups.

In certain embodiments, the GLS-1 inhibitor is of Formula IIIc:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

In certain embodiments, the GLS1i is of Formula IIIc as shown above, ora salt thereof, wherein R¹ is methyl.

In certain embodiments, the GLS-1 inhibitor is of Formula IIIc-1:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

In certain embodiments, the GLS-1 inhibitor is of Formula IIIc-2:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R¹ is chosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups;

each R⁴ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,and hydroxyl, wherein R⁴ may be optionally substituted with one to threeR^(Z) groups;

each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; and

each R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.

In certain embodiments, the GLS-1 inhibitor is of Formula IIId:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

each of R^(Z1) and R^(Z2) is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, and oxo.

In certain embodiments, the GLS-1 inhibitor is of Formula IIId as shownabove, or a salt thereof, wherein:

R^(X) is chosen from fluoro and H; and

each of R^(Z1) and R^(Z2) is independently chosen from alkyl,cycloalkyl, cycloalkylhaloalkyl, cycloalkyloxy, H, haloalkoxy,haloalkoxyaryl, haloalkyl, halocycloalkyloxy, heterocycloalkyl, andheterocycloalkyloxy.

In certain embodiments, the GLS-1 inhibitor is of Formula IIId as shownabove, or a salt thereof, wherein:

R^(X) is chosen from fluoro and H; and

each of R^(Z1) and R^(Z2) is independently chosen from H,

In certain embodiments, the GLS-1 inhibitor is of Formula IIIe:

or a salt thereof, wherein:

R^(X) is chosen from fluoro and H;

R^(Z1) is chosen from alkenyl, alkoxy, alkoxyalkyl, alkoxyaryl,alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, and oxo.

In certain embodiments, the GLS-1 inhibitor is a compound or a saltthereof, wherein the compound is chosen from

In certain embodiments, the GLS1i is one as disclosed in United StatesPatent Application Publication No. US 2017/0001996 filed Jan. 5, 2017.

In certain embodiments, the GLS-1 inhibitor is of Formula IV:

or a salt thereof, wherein:

n is chosen from 1 and 2;

R¹ is chosen from NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂, C(O)N(R³)₂, andN(R³)₂;

each R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(x) groups, wherein two R³groups together with the atoms to which they are attached optionallyform an heteroaryl or heterocycloalkyl ring, which may be optionallysubstituted with one to three R^(x) groups;

R² is chosen from NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂, C(O)N(R⁴)₂ andN(R⁴)₂;

each R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups, wherein two R⁴groups together with the atoms to which they are attached optionallyform an heteroaryl or heterocycloalkyl ring, which may be optionallysubstituted with one to three R′ groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(Z) groups;

R^(Z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl;

A is a monocyclic heteroaryl, which may be optionally substituted withone to three R^(Z) groups; and

Z is a monocyclic heteroaryl, which may be optionally substituted withone to three R^(Z) groups.

In certain embodiments, the GLS1i is disclosed in United States PatentApplication Publication No. US 2017/0001996, published Jan. 5, 2017.

In certain embodiments, the GLS-1 inhibitor is of Formula IVa:

or a salt thereof, wherein:

n is chosen from 1 and 2;

A¹ is chosen from S and HC═CH;

Z¹ is chosen from S, CH, and HC═CH;

Z² is N when Z¹ is CH, and Z² is C when Z¹ is S or HC═CH;

R¹ is chosen from NR³C(O)R³, NR³C(O)OR³, NR³C(O)N(R³)₂, C(O)N(R³)₂, andN(R³)₂;

each R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(X) groups, wherein two R³groups together with the atoms to which they are attached optionallyform an heteroaryl or heterocycloalkyl ring, which may be optionallysubstituted with one to three R^(X) groups;

R² is chosen from NR⁴C(O)R⁴, NR⁴C(O)OR⁴, NR⁴C(O)N(R⁴)₂, C(O)N(R⁴)₂ andN(R⁴)₂;

each R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups, wherein two R⁴groups together with the atoms to which they are attached optionallyform an heteroaryl or heterocycloalkyl ring, which may be optionallysubstituted with one to three R^(x) groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(z) groups; and

R^(z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl.

In certain embodiments, the GLS-1 inhibitor is of Formula IVb:

or a salt thereof, wherein:

n is chosen from 1 and 2;

R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(x) groups;

R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(z) groups; and

R^(z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl.

In certain embodiments, the GLS-1 inhibitor is of Formula IVc:

or a salt thereof, wherein:

n is chosen from 1 and 2;

R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(x) groups;

R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(z) groups; and

R^(z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl.

In certain embodiments, the GLS-1 inhibitor is of Formula IVd:

or a salt thereof, wherein:

n is chosen from 1 and 2;

R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(x) groups;

R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(Z) groups; and

R^(z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl.

In certain embodiments, the GLS-1 inhibitor is of Formula IVe:

or a salt thereof, wherein:

n is chosen from 1 and 2;

R³ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R³ may beoptionally substituted with one to three R^(X) groups;

R⁴ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein each R⁴ may beoptionally substituted with one to three R^(x) groups;

each R^(x) group is independently chosen from alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, C(O)N(R⁵)₂, and C(O)R⁵;

each R⁵ is independently chosen from alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, which may be optionallysubstituted with one to three R^(z) groups; and

R^(z) is chosen from alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl.

In certain embodiments, the GLS1i is one as disclosed in US2017/0174661, published Jun. 22, 2017.

In certain embodiments, the GLS1i is one as disclosed in WO2013/078123or Mol Cancer Ther 2014; 13:890-901.

In certain embodiments, the GLS-1 inhibitor is of Formula A1,

or a pharmaceutically acceptable salt thereof, wherein:

L represents CH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂,CH═CH, or cyclopropyl, preferably CH₂CH₂, wherein any hydrogen atom of aCH or CH₂ unit may be replaced by alkyl or alkoxy, any hydrogen of an NHunit may be replaced by alkyl, and any hydrogen atom of a CH₂ unit ofCH₂CH₂, CH₂CH₂CH₂ or CH₂ may be replaced by hydroxy;

X, independently for each occurrence, represents S, O or CH═CH,preferably S or CH═CH, wherein any hydrogen atom of a CH unit may bereplaced by alkyl;

Y, independently for each occurrence, represents H or CH₂O(CO)R₇;

R₇, independently for each occurrence, represents H or substituted orunsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;

Z represents H or R₃(CO);

R₁ and R₂ each independently represent H, alkyl, alkoxy or hydroxy; R₃,independently for each occurrence, represents substituted orunsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl,alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀),N(R₄)(R₅) or OR₆, wherein any free hydroxyl group may be acylated toform C(O)R₇;

R₄ and R₅ each independently represent H or substituted or unsubstitutedalkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl,alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any freehydroxyl group may be acylated to form C(O)R₇;

R₆, independently for each occurrence, represents substituted orunsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl,alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any freehydroxyl group may be acylated to form C(O)R₇; and

R₈, R₉ and R₁₀ each independently represent H or substituted orunsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, or R₈and R₉ together with the carbon to which they are attached, form acarbocyclic or heterocyclic ring system, wherein any free hydroxyl groupmay be acylated to form C(O)R₇, and wherein at least two of R₈, R₉ andR₁₀ are not H.

In certain embodiments, the GLS1i is one as disclosed in WO2014/078645.

In certain embodiments, the GLS-1 inhibitor is of Formula A2,

or a pharmaceutically acceptable salt thereof, wherein:

L represents CH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂,CH═CH, or cyclopropyl, preferably CH₂CH₂, wherein any hydrogen atom of aCH or CH₂ unit may be replaced by alkyl or alkoxy, any hydrogen of an NHunit may be replaced by alkyl, and any hydrogen atom of a CH₂ unit ofCH₂CH₂, CH₂CH₂CH₂ or CH₂ may be replaced by hydroxy;

X represents S, O or CH═CH, preferably S or CH═CH, wherein any hydrogenatom of a CH unit may be replaced by alkyl;

Y, independently for each occurrence, represents H or CH₂O(CO)R₇;

R₇, independently for each occurrence, represents H or substituted orunsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;

Z represents H or R₃(CO);

R₁ and R₂ each independently represent H, alkyl, alkoxy or hydroxy;

R₃ represents substituted or unsubstituted alkyl, hydroxyalkyl,aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀), N(R₄)(R₅) or OR₆,wherein any free hydroxyl group may be acylated to form C(O)R₇;

R₄ and R₅ each independently for each occurrence represent H orsubstituted or unsubstituted alkyl, hydroxyalkyl, acyl, aminoalkyl,acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy,aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, orheteroaryloxyalkyl, wherein any free hydroxyl group may be acylated toform C(O)R₇;

R₆ represents substituted or unsubstituted alkyl, hydroxyalkyl,aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl,aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, orheteroaryloxyalkyl, wherein any free hydroxyl group may be acylated toform C(O)R₇;

R₈, R₉ and R₁₀ each independently for each occurrence represent H orsubstituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino,acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl,alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl,aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, orheteroaryloxyalkyl, or R8 and R9 together with the carbon to which theyare attached, form a carbocyclic or heterocyclic ring system, whereinany free hydroxyl group may be acylated to form C(O)R₇, and wherein atleast two of R₈, R₉ and R₁₀ are not H;

R₁₁ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the arylor heteroaryl ring is substituted with either —OCHF₂ or —OCF₃ and isoptionally further substituted, or R₁₁ represents C(R₁₂)(R₁₃)(R₁₄),N(R₄)(R₁₄) or OR₁₄, wherein any free hydroxyl group may be acylated toform C(O)R₇;

R₁₂ and R₁₃ each independently represent H or substituted orunsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,wherein any free hydroxyl group may be acylated to form C(O)R₇, andwherein both of R₁₂ and R₁₃ are not H; and

R₁₄ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the arylor heteroaryl ring is substituted with either —OCHF₂ or —OCF₃ and isoptionally further substituted.

In certain embodiments, the GLS1i is one as disclosed in WO2014/079011.

In certain embodiments, the GLS-1 inhibitor is of Formula A3,

or a pharmaceutically acceptable salt thereof, wherein:

X is C₃-C₇ cycloalkylene;

each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, providedthat at least one of W, Y and Z is not —CH═;

each R₁ and R₂ is independently —NH₂, —N(R₃)—C(O)—R₄, —C(O)—N(R₃)—R₄,N(R₃)—C(O)—O—R₄, —N(R₃)—C(O)—N(R₃)—R₄ or —N(R₃)—C(O)—SR₄;

each R₃ is independently hydrogen, C₁₋₆ alkyl or aryl;

each R₄ is independently C₁₋₆ alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, orheterocyclyl, each of which is substituted with 0-3 occurrences of R₅;

each R₁ is independently C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ thioalkoxy, C₁₋₆haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkylalkyl, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cyano, halo,oxo, —OH, —OCF₃, —SO₂—C₁₋₆ alkyl, —NO₂, —N(R₇)—C(O)—C₁₋₆ alkyl, —N(R₇)₂,or two adjacent R₅ moieties, taken together with the atoms to which theyare attached form a heterocyclyl;

each R₆ is independently hydrogen, fluoro, C₁₋₆ alkyl, —OH, —NH₂,—NH(CH₃), N(CH₃)₂, or C₁₋₆ alkoxy;

each R₇ is independently hydrogen or C₁₋₆ alkyl;

m is 0, 1, or 2;

n is 0, 1, or 2;

o is 1, 2 or 3; and

p is 1, 2 or 3.

In certain embodiments, the GLS1i is one as disclosed in WO2014/081925A1or US2014/0142081A1.

In certain embodiments, the GLS-1 inhibitor is of Formula A4,

or a pharmaceutically acceptable salt thereof, wherein:

X is a bond —S—, —S(O)—, —SO₂—, —CH═CH—, —NH— or —C(O)—;

each W, Y and Z is independently —S—, —CH═, —CH═CH—, —CH═CR₁—,—CR₁═CR₁—, —O—, —N═, or —NH—, provided that (1) for each ring at leastone of W, Y and Z is not-CH═ and (2) when one of W is-S— and the Y inthe same ring is N, then the Z in the same ring is not-CH═;

each R₁ and R₂ is independently C₁₋₆ alkylene-R₄, —N(R₃)—R₄,—N(R₃)—C(O)—R₄, C(O)—N(R₃)—R₄, —N(R₃)—C(O)—O—R₄, —N(R₃)—C(O)—N(R₃)—R₄,—O—C(O)—N(R₃)—R₄, —N(R₃)C(O)—C₁₋₆ alkylene-C(O)—R₄, —N(R₃)—C(O)—C₁₋₆alkylene-N(R₃)—C(O)—R₄ or —N(R_(3a))—C(O)CH₂—N(R₃)—C(O)—R₄;

each R₃ and R_(3a) is independently hydrogen, C₁₋₆ alkyl or aryl;

each R₄ is independently C₁₋₆ alkyl, C₁₋₆ alkenyl, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cycloalkyl orcycloalkylalkyl, each of which is substituted with 0-3 occurrences ofR₅, or two adjacent R₅ moieties, taken together with the atoms to whichthey are attached form a heterocyclyl, heteroaryl, cycloalkyl or aryl;

each R₅ is independently oxo (═O), C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, cyano, halo, —OH, —SH, —OCF₃, —SO₂C₁₋₆ alkyl, —NO₂,—N(R₇)—C(O)—C₁₋₆ alkyl, —N(R₆)₂, —O—C(O)C₁₋₆ alkyl, C₃₋₇ cycloalkyl,(C₃₋₇ cycloalkyl)alkyl, aryl, aryloxy, —C(O)-aryl, heteroaryl, aralkyl,heteroaralkyl, heterocyclylalkyl or heterocyclyl, wherein each aryl,heteroaryl or heterocyclyl is further substituted with 0-3 occurrencesof R₇;

each R₆ is independently hydrogen, fluoro, OH or C₁₋₆ alkyl;

each R₇ is independently hydrogen, C₁₋₆ alkyl, —OH, —SH, cyano, halo,—CF₃, OCF₃, —SO₂—C₁₋₆ alkyl, —NO₂, —N(R₇)—C(O)—C₁₋₆ alkyl, —N(R₆)₂ orC₁₋₆ alkoxy;

m is 1, 2 or 3;

n is 1, 2 or 3; provided that when X is bond, the sum of m and n is from3 to 6 and when X is —S—, —S(O)—, —SO₂, —CH═CH—, or —C(O)—, the sum of mand n is from 2 to 4;

o is 1, 2 or 3; and

p is 1, 2 or 3.

Pharmaceutical Compositions

While it may be possible for the compounds disclosed herein to beadministered as the raw chemical, it is also possible to present them asa pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein may be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject disclosure or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Routes of Administration Oral Administration

The compounds of the present invention may be administered orally,including swallowing, so the compound enters the gastrointestinal tract,or is absorbed into the blood stream directly from the mouth, includingsublingual or buccal administration.

Suitable compositions for oral administration include solid formulationssuch as tablets, pills, cachets, lozenges and hard or soft capsules,which can contain liquids, gels, powders, or granules.

In a tablet or capsule dosage form the amount of drug present may befrom about 0.05% to about 95% by weight, more typically from about 2% toabout 50% by weight of the dosage form.

In addition, tablets or capsules may contain a disintegrant, comprisingfrom about 0.5% to about 35% by weight, more typically from about 2% toabout 25% of the dosage form. Examples of disintegrants include methylcellulose, sodium or calcium carboxymethyl cellulose, croscarmellosesodium, polyvinylpyrrolidone, hydroxypropyl cellulose, starch and thelike.

Suitable binders, for use in a tablet, include gelatin, polyethyleneglycol, sugars, gums, starch, hydroxypropyl cellulose and the like.Suitable diluents, for use in a tablet, include mannitol, xylitol,lactose, dextrose, sucrose, sorbitol and starch.

Suitable surface active agents and glidants, for use in a tablet orcapsule, may be present in amounts from about 0.1% to about 3% byweight, and include polysorbate 80, sodium dodecyl sulfate, talc andsilicon dioxide.

Suitable lubricants, for use in a tablet or capsule, may be present inamounts from about 0.1% to about 5% by weight, and include calcium, zincor magnesium stearate, sodium stearyl fumarate and the like.

Tablets may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed withbinders, inert diluents, or lubricating, surface active or dispersingagents. Molded tablets may be made by molding in a suitable machine amixture of the powdered compound moistened with a liquid diluent. Dyesor pigments may be added to tablets for identification or tocharacterize different combinations of active compound doses.

Liquid formulations can include emulsions, solutions, syrups, elixirsand suspensions, which can be used in soft or hard capsules. Suchformulations may include a pharmaceutically acceptable carrier, forexample, water, ethanol, polyethylene glycol, cellulose, or an oil. Theformulation may also include one or more emulsifying agents and/orsuspending agents.

Compositions for oral administration may be formulated as immediate ormodified release, including delayed or sustained release, optionallywith enteric coating.

In another embodiment, a pharmaceutical composition comprises atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Parenteral Administration

Compounds of the present invention may be administered directly into theblood stream, muscle, or internal organs by injection, e.g., by bolusinjection or continuous infusion. Suitable means for parenteraladministration include intravenous, intra-muscular, subcutaneousintraarterial, intraperitoneal, intrathecal, intracranial, and the like.Suitable devices for parenteral administration include injectors(including needle and needle-free injectors) and infusion methods. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials.

Most parenteral formulations are aqueous solutions containingexcipients, including salts, buffering, suspending, stabilizing and/ordispersing agents, antioxidants, bacteriostats, preservatives, andsolutes which render the formulation isotonic with the blood of theintended recipient, and carbohydrates.

Parenteral formulations may also be prepared in a dehydrated form (e.g.,by lyophilization) or as sterile non-aqueous solutions. Theseformulations can be used with a suitable vehicle, such as sterile water.Solubility-enhancing agents may also be used in preparation ofparenteral solutions.

Compositions for parenteral administration may be formulated asimmediate or modified release, including delayed or sustained release.Compounds may also be formulated as depot preparations. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Topical Administration

Compounds of the present invention may be administered topically (forexample to the skin, mucous membranes, ear, nose, or eye) ortransdermally. Formulations for topical administration can include, butare not limited to, lotions, solutions, creams, gels, hydrogels,ointments, foams, implants, patches and the like. Carriers that arepharmaceutically acceptable for topical administration formulations caninclude water, alcohol, mineral oil, glycerin, polyethylene glycol andthe like. Topical administration can also be performed by, for example,electroporation, iontophoresis, phonophoresis and the like.

Typically, the active ingredient for topical administration may comprisefrom 0.001% to 10% w/w (by weight) of the formulation. In certainembodiments, the active ingredient may comprise as much as 10% w/w; lessthan 5% w/w; from 2% w/w to 5% w/w; or from 0.1% to 1% w/w of theformulation.

Compositions for topical administration may be formulated as immediateor modified release, including delayed or sustained release.

Rectal, Buccal, and Sublingual Administration

Suppositories for rectal administration of the compounds of the presentinvention can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di-, ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the drug.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

Administration by Inhalation

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray or powder. Pressurizedpacks may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Alternatively, foradministration by inhalation or insufflation, the compounds according tothe disclosure may take the form of a dry powder composition, forexample a powder mix of the compound and a suitable powder base such aslactose or starch. The powder composition may be presented in unitdosage form, in for example, capsules, cartridges, gelatin or blisterpacks from which the powder may be administered with the aid of aninhalator or insufflator.

Other carrier materials and modes of administration known in thepharmaceutical art may also be used. Pharmaceutical compositions of theinvention may be prepared by any of the well-known techniques ofpharmacy, such as effective formulation and administration procedures.Preferred unit dosage formulations are those containing an effectivedose, as herein recited, or an appropriate fraction thereof, of theactive ingredient. The precise amount of compound administered to apatient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. In addition, the route ofadministration may vary depending on the condition and its severity. Theabove considerations concerning effective formulations andadministration procedures are well known in the art and are described instandard textbooks. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et al., Eds.,Handbook of Pharmaceutical Excipients (3^(rd) Ed.), AmericanPharmaceutical Association, Washington, 1999.

Methods of Treatment

The methods of the invention can be used to treat any subject in need oftreatment. Examples of subjects or patients include, but are not limitedto, humans, monkeys, deer, camel, pets and companion animals, including,but not limited to, dogs, cats, horses, rabbits, and guinea pigs;livestock, including, but not limited to, cows, buffalo, bison, mules,goats, sheep and pigs. In one embodiment, the subject is a human.

The methods of the invention provide for the administration of aglutaminase inhibitor or a compound that inhibits glutathione productionfor the treatment of several diseases and disorders. The glutaminaseinhibitor may, for example, be a GLS-1 inhibitor or a selectiveinhibitor of GLS-1. In one embodiment, the glutaminase inhibitor iscompound 1, compound 2 or a compound selected from Table 1.

In one embodiment the disorder is a cancer, including, but not limitedto, bladder cancer, bone marrow cancer, breast cancer, cancer of thecentral nervous system, cervical cancer, colon cancer, endometrialcancer, cancer of the gastric system, head and neck cancer, kidneycancer, leukemia, liver cancer, lung cancer, lymphoma, muscle cancer,ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroidcancer, or a variant thereof. In another embodiment, the disorder isovarian cancer, including, but not limited to, high-grade serous ovariancancer (HGSOC) or nonresectable or relapsed HGSOC.

In one embodiment, provided herein is a method of treatment of a diseaseor disorder comprising the administration of a glutaminase inhibitor ora compound that inhibits glutathione production. Examples of diseases ordisorders include, but are not limited to, cancers, as provided above.Examples of glutaminase inhibitors include, but are not limited to,GLS-1 inhibitors or a selective inhibitor of GLS-1 as provided above.

In another embodiment, provided herein is a glutaminase inhibitor or acompound that inhibits glutathione production for use as a medicament.For example, provided herein is a glutaminase inhibitor or a compoundthat inhibits glutathione production for use as a medicament in thetreatment of a disease or disorder, including, but not limited to, thetreatment of various cancers. In certain embodiments, the cancer isovarian cancer, including, but not limited to, HGSOC or nonresectable orrelapsed HGSOC. The glutaminase inhibitor may, for example, be a GLS-1inhibitor or a selective inhibitor of GLS-1 as provided above.

In yet another embodiment, provided herein is the use of a glutaminaseinhibitor or a compound that inhibits glutathione production for use inthe manufacture of a medicament. For example, provided herein is the useof a glutaminase inhibitor or a compound that inhibits glutathioneproduction for use in the manufacture of a medicament for the treatmentof a disease or disorder, including, but not limited to, the treatmentof various cancers. In certain embodiments, the cancer is ovariancancer, including, but not limited to, HGSOC or nonresectable orrelapsed HGSOC. The glutaminase inhibitor may, for example, be a GLS-1inhibitor or a selective inhibitor of GLS-1. In one embodiment, theglutaminase inhibitor is compound 1, compound 2 or a compound selectedfrom Table 1.

In some embodiments, the cancer is bladder cancer, bone marrow cancer,breast cancer, cancer of the central nervous system, cervical cancer,colon cancer, endometrial cancer, cancer of the gastric system, head andneck cancer, kidney cancer, leukemia, liver cancer, lung cancer,lymphoma, muscle cancer, ovarian cancer, pancreatic cancer, prostatecancer, skin cancer, thyroid cancer, or a variant thereof. In certainembodiments, the cancer is ovarian cancer, including, but not limitedto, HGSOC.

In some embodiments, methods described herein are used to treat adisease or condition comprising administering to a subject in needthereof a therapeutically effective amount of a GLS-1 inhibitor orpharmaceutically acceptable salt thereof, wherein the condition iscancer which has developed resistance to chemotherapeutic drugs and/orionizing radiation. In certain embodiments, the cancer is ovariancancer, including, but not limited to, HGSOC or nonresectable orrelapsed HGSOC. The glutaminase inhibitor may, for example, be a GLS-1inhibitor or a selective inhibitor of GLS-1. In one embodiment, theglutaminase inhibitor is compound 1, compound 2 or a compound selectedfrom Table 1.

Combinations and Combination Therapy

The compounds of the present invention can be used, alone or incombination with other pharmaceutically active compounds, to treatconditions such as those disclosed hereinabove. The compound(s) of thepresent invention and other pharmaceutically active compound(s) can beadministered simultaneously (either in the same dosage form or inseparate dosage forms) or sequentially. Accordingly, in one embodiment,the present invention comprises methods for treating a condition byadministering to the subject a therapeutically-effective amount of oneor more compounds of the present invention and one or more additionalpharmaceutically active compounds.

In another embodiment, provided herein is a glutaminase inhibitor or acompound that inhibits glutathione production for use as a medicament incombination with one or more additional pharmaceutically activecompounds. In yet another embodiment, provided herein is the use of aglutaminase inhibitor or a compound that inhibits glutathione productionand one or more additional pharmaceutically active compounds for themanufacturing of a medicament. For example, provided herein is the useof a glutaminase inhibitor or a compound that inhibits glutathioneproduction and one or more additional pharmaceutically active compoundsfor the manufacturing of a medicament for the treatment of a disease ordisorder, including, but not limited to, the treatment of variouscancers.

In another embodiment, there is provided a pharmaceutical compositioncomprising one or more compounds of the present invention, one or moreadditional pharmaceutically active compounds, and a pharmaceuticallyacceptable carrier.

In another embodiment, the one or more additional pharmaceuticallyactive compounds is chosen from anti-cancer drugs, anti-proliferativedrugs, and anti-inflammatory drugs. In certain embodiments, theanti-cancer agent is chosen from a platinum-based agent, a taxane-basedagent, an immunotherapy, and a targeted therapy. In certain embodiments,the targeted therapy is an inhibitor of MEK kinase, HSP90, CDK4, or themTOR pathway.

Glutaminase inhibitors, e.g., GLS-1 inhibitors, disclosed herein arealso optionally used in combination with other therapeutic reagents thatare selected for their therapeutic value for the condition to betreated. In general, the compounds described herein and, in embodimentswhere combination therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition and, because ofdifferent physical and chemical characteristics, are optionallyadministered by different routes. The initial administration isgenerally made according to established protocols and then, based uponthe observed effects, the dosage, modes of administration and times ofadministration subsequently modified. In certain instances, it isappropriate to administer a glutaminase inhibitor compound, as disclosedherein, in combination with another therapeutic agent. By way of exampleonly, the therapeutic effectiveness of a glutaminase inhibitor isenhanced by administration of another therapeutic agent (which alsoincludes a therapeutic regimen) that also has therapeutic benefit.Regardless of the disease, disorder or condition being treated, theoverall benefit experienced by the patient is either simply additive ofthe two therapeutic agents or the patient experiences an enhanced (i.e.,synergistic) benefit. Alternatively, if a compound disclosed herein hasa side effect, it may be appropriate to administer an agent to reducethe side effect; or the therapeutic effectiveness of a compounddescribed herein may be enhanced by administration of an adjuvant.

Therapeutically effective dosages vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically effective dosages of drugs and other agents for use incombination treatment regimens are documented methodologies. Combinationtreatment further includes periodic treatments that start and stop atvarious times to assist with the clinical management of the patient. Inany case, the multiple therapeutic agents (one of which is a glutaminaseinhibitor, e.g., a GLS-1 inhibitor, as disclosed herein) may beadministered in any order, or simultaneously. If simultaneously, themultiple therapeutic agents are optionally provided in a single, unifiedform, or in multiple forms (by way of example only, either as a singlepill or as two separate pills).

In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses optionally varies from more than zeroweeks to less than twelve weeks.

In addition, the combination methods, compositions and formulations arenot to be limited to the use of only two agents, the use of multipletherapeutic combinations are also envisioned. It is understood that thedosage regimen to treat, prevent, or ameliorate the condition(s) forwhich relief is sought, is optionally modified in accordance with avariety of factors. These factors include the disorder from which thesubject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, the dosage regimen actually employedvaries widely, in some embodiments, and therefore deviates from thedosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein are optionally a combined dosage form or in separatedosage forms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy areoptionally also administered sequentially, with either agent beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen optionally calls for sequentialadministration of the active agents or spaced-apart administration ofthe separate active agents. The time between the multiple administrationsteps ranges from a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent.

In another embodiment, a glutaminase inhibitor, e.g., a GLS1i, isoptionally used in combination with procedures that provide additionalbenefit to the patient. A glutaminase inhibitor and any additionaltherapies are optionally administered before, during, or after theoccurrence of a disease or condition, and the timing of administeringthe composition containing a glutaminase inhibitor, e.g., a GLS1i,varies in some embodiments. Thus, for example, a glutaminase inhibitoris used as a prophylactic and is administered continuously to subjectswith a propensity to develop conditions or diseases in order to preventthe occurrence of the disease or condition. A glutaminase inhibitor,e.g., a GLS1i, and compositions are optionally administered to a subjectduring or as soon as possible after the onset of the symptoms. Whileembodiments of the present invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood that insome embodiments of the invention various alternatives to theembodiments described herein are employed in practicing the invention.

A glutaminase inhibitor, e.g., a GLS1i, disclosed herein can be used incombination with anti-cancer drugs, including but not limited to thefollowing classes: alkylating agents, anti-metabolites, plant alkaloidsand terpenoids, topoisomerase inhibitors, cytotoxic antibiotics,angiogenesis inhibitors and tyrosine kinase inhibitors.

For use in the treatment or attenuation of cancer and neoplasticdiseases a glutaminase inhibitor may be optimally used together with oneor more of the following non-limiting examples of anti-cancer agents,including, but not limited to:

-   -   1) inhibitors or modulators of a protein involved in one or more        of the DNA damage repair (DDR) pathways such as:        -   a. PARP1/2, including, but not limited to: olaparib,            niraparib, rucaparib;        -   b. checkpoint kinase 1 (CHK1), including, but not limited            to: UCN-01, AZD7762, PF477736, SCH900776, MK-8776,            LY2603618, V158411, and EXEL-9844;        -   c. checkpoint kinase 2 (CHK2), including, but not limited            to: PV1019, NSC 109555, and VRX0466617;        -   d. dual CHK1/CHK2, including, but not limited to: XL-844,            AZD7762, and PF-473336;        -   e. WEE1, including, but not limited to: MK-1775 and            PD0166285;        -   f. ATM, including, but not limited to KU-55933,        -   g. DNA-dependent protein kinase, including, but not limited            to NU7441 and M3814; and        -   h. Additional proteins involved in DDR;    -   2) Inhibitors or modulators of one or more immune checkpoints,        including, but not limited to:        -   a. PD-1 inhibitors such as nivolumab (OPDIVO), pembrolizumab            (KEYTRUDA), pidilizumab (CT-011), and AMP-224 (AMPLIMMUNE);    -   b. PD-L1 inhibitors such as Atezolizumab (TECENTRIQ), Avelumab        (Bavencio), Durvalumab (Imfinzi), MPDL3280A (Tecentriq),        BMS-936559, and MEDI4736;        -   c. anti-CTLA-4 antibodies such as ipilimumab (YERVOY) and            CP-675,206 (TREMELIMUMAB);        -   d. inhibitors of T-cell immunoglobulin and mucin domain 3            (Tim-3);        -   e. inhibitors of V-domain Ig suppressor of T cell activation            (Vista);        -   f. inhibitors of band T lymphocyte attenuator (BTLA);        -   g. inhibitors of lymphocyte activation gene 3 (LAG3); and        -   h. inhibitors of T cell immunoglobulin and immunoreceptor            tyrosine-based inhibitory motif domain (TIGIT);    -   3) telomerase inhibitors or telomeric DNA binding compounds;    -   4) alkylating agents, including, but not limited to:        chlorambucil (LEUKERAN), oxaliplatin (ELOXATIN), streptozocin        (ZANOSAR), dacarbazine, ifosfamide, lomustine (CCNU),        procarbazine (MATULAN), temozolomide (TEMODAR), and thiotepa;    -   5) DNA crosslinking agents, including, but not limited to:        carmustine, chlorambucil (LEUKERAN), carboplatin (PARAPLATIN),        cisplatin (PLATIN), busulfan (MYLERAN), melphalan (ALKERAN),        mitomycin (MITOSOL), and cyclophosphamide (ENDOXAN);    -   6) anti-metabolites, including, but not limited to: cladribine        (LEUSTATIN), cytarbine, (ARA-C), mercaptopurine (PURINETHOL),        thioguanine, pentostatin (NIPENT), cytosine arabinoside        (cytarabine, ARA-C), gemcitabine (GEMZAR), fluorouracil (5-FU,        CARAC), capecitabine (XELODA), leucovorin (FUSILEV),        methotrexate (RHEUMATREX), and raltitrexed;    -   7) antimitotics, which are often plant alkaloids and terpenoids,        or derivateves thereof including but limited to: taxanes such as        docetaxel (TAXITERE), paclitaxel (ABRAXANE, TAXOL), vinca        alkaloids such as vincristine (ONCOVIN), vinblastine, vindesine,        and vinorelbine (NAVELBINE);    -   8) topoisomerase inhibitors, including, but not limited to:        amsacrine, camptothecin (CTP), genisten, irinotecan (CAMPTOSAR),        topotecan (HYCAMTIN), doxorubicin (ADRIAMYCIN), daunorubicin        (CERUBIDINE), epirubicin (ELLENCE), ICRF-193, teniposide        (VUMON), mitoxantrone (NOVANTRONE), and etoposide (EPOSIN);    -   9) DNA replication inhibitors, including, but not limited to:        fludarabine (FLUDARA), aphidicolin, ganciclovir, and cidofovir;    -   10) ribonucleoside diphosphate reductase inhibitors, including,        but not limited to: hydroxyurea;    -   11) transcription inhibitors, including, but not limited to:        actinomycin D (dactinomycin, COSMEGEN) and plicamycin        (mithramycin);    -   12) DNA cleaving agents, including, but not limited to:        bleomycin (BLENOXANE), idarubicin,    -   13) cytotoxic antibiotics, including, but not limited to:        actinomycin D (dactinomycin, COSMEGEN),    -   14) aromatase inhibitors, including, but not limited to:        aminoglutethimide, anastrozole (ARIMIDEX), letrozole (FEMARA),        vorozole (RIVIZOR), and exemestane (AROMASIN);    -   15) angiogenesis inhibitors, including, but not limited to:        genistein, sunitinib (SUTENT), and bevacizumab (AVASTIN);    -   16) anti-steroids and anti-androgens, including, but not limited        to: aminoglutethimide (CYTADREN), bicalutamide (CASODEX),        cyproterone, flutamide (EULEXIN), nilutamide (NILANDRON);    -   17) tyrosine kinase inhibitors, including, but not limited to:        imatinib (GLEEVEC), erlotinib (TARCEVA), lapatininb (TYKERB),        sorafenib (NEXAVAR), and axitinib (INLYTA);    -   18) mTOR inhibitors, including, but not limited to: everolimus,        temsirolimus (TORISEL), and sirolimus;    -   19) monoclonal antibodies, including, but not limited to:        trastuzumab (HERCEPTIN) and rituximab (RITUXAN);    -   20) apoptosis inducers such as cordycepin;    -   21) protein synthesis inhibitors, including, but not limited to:        clindamycin, chloramphenicol, streptomycin, anisomycin, and        cycloheximide;    -   22) antidiabetics, including, but not limited to: metformin and        phenformin;    -   23) antibiotics, including, but not limited to:        -   a. tetracyclines, including, but not limited to:            doxycycline;        -   b. erythromycins, including, but not limited to:            azithromycin;        -   c. glycylglycines, including, but not limited to:            tigecyline;        -   d. antiparasitics, including, but not limted to: pyrvinium            pamoate;        -   e. beta-lactams, including, but not limited to the            penicillins and cephalosporins;        -   f. anthracycline antibiotics, including, but not limited to:            daunorubicin and doxorubicin;        -   g. other antibiotics, including, but not limited to:            chloramphenicol, mitomycin C, and actinomycin;    -   24) antibody therapeutical agents, including, but not limited        to: muromonab-CD3, infliximab (REMICADE), adalimumab (HUMIRA),        omalizumab (XOLAIR), daclizumab (ZENAPAX), rituximab (RITUXAN),        ibritumomab (ZEVALIN), tositumomab (BEXXAR), cetuximab        (ERBITUX), trastuzumab (HERCEPTIN), ADCETRIS, alemtuzumab        (CAMPATH-1H), Lym-1 (ONCOLYM), ipilimumab (YERVOY), vitaxin,        bevacizumab (AVASTIN), and abciximab (REOPRO); and    -   25) other agents, such as Bacillus Calmette-Guérin (B-C-G)        vaccine; buserelin (ETILAMIDE); chloroquine (ARALEN);        clodronate, pamidronate, and other bisphosphonates; colchicine;        demethoxyviridin; dichloroacetate; estramustine; filgrastim        (NEUPOGEN); fludrocortisone (FLORINEF); goserelin (ZOLADEX);        interferon; leucovorin; leuprolide (LUPRON); levamisole;        lonidamine; mesna; metformin; mitotane (o,p′-DDD, LYS ODREN);        nocodazole; octreotide (SANDOSTATIN); perifosine; porfimer        (particularly in combination with photo- and radiotherapy);        suramin; tamoxifen; titanocene dichloride; tretinoin; anabolic        steroids such as fluoxymesterone (HALOTESTIN); estrogens such as        estradiol, diethylstilbestrol (DES), and dienestrol; progestins        such as medroxyprogesterone acetate (MPA) and megestrol; and        testosterone;

Where a subject is suffering from or at risk of suffering from aninflammatory condition, a glutaminase inhibitor, e.g., a GLS1i, compounddisclosed herein is optionally used together with one or more agents ormethods for treating an inflammatory condition in any combination.Therapeutic agents/treatments for treating an autoimmune and/orinflammatory condition include, but are not limited to any of thefollowing examples:

-   -   1) corticosteroids, including but not limited to cortisone,        dexamethasone, and methylprednisolone;    -   2) nonsteroidal anti-inflammatory drugs (NSAIDs), including but        not limited to ibuprofen, naproxen, acetaminophen, aspirin,        fenoprofen (NALFON), flurbiprofen (ANSAID), ketoprofen,        oxaprozin (DAYPRO), diclofenac sodium (VOLTAREN), diclofenac        potassium (CATAFLAM), etodolac (LODINE), indomethacin (INDOCIN),        ketorolac (TORADOL), sulindac (CLINORIL), tolmetin (TOLECTIN),        meclofenamate (MECLOMEN), mefenamic acid (PONSTEL), nabumetone        (RELAFEN) and piroxicam (FELDENE);    -   3) immunosuppressants, including but not limited to methotrexate        (RHEUMATREX), leflunomide (ARAVA), azathioprine (IMURAN),        cyclosporine (NEORAL, SANDIMMUNE), tacrolimus and        cyclophosphamide (CYTOXAN);    -   4) CD20 blockers, including but not limited to rituximab        (RITUXAN);    -   5) Tumor Necrosis Factor (TNF) blockers, including but not        limited to etanercept (ENBREL), infliximab (REMICADE) and        adalimumab (HUMIRA);    -   6) interleukin-1 receptor antagonists, including but not limited        to anakinra (KINERET);    -   7) interleukin-6 inhibitors, including but not limited to        tocilizumab (ACTEMRA);    -   8) interleukin-17 inhibitors, including but not limited to        AIN457;    -   9) Janus kinase inhibitors, including but not limited to        tasocitinib; and    -   10) syk inhibitors, including but not limited to fostamatinib.        General Synthetic Methods for Preparing Compounds

Compounds useful in the methods of the present invention can be preparedusing methods that are known to one of skill in the art. Startingmaterials used to prepare compounds of the present invention arecommercially available or can be prepared using routine methods known inthe art.

Biological Assays

The following are examples of biological assays useful with the methodsof the invention. The assays provided herein are not limiting and otherassays now known, or later discovered, by one of skill in the art can beused for the same purpose as the assays provided below.

Compounds disclosed herein are active as GLS-1 inhibitors. Certaincompounds disclosed in Table 1 were synthesized and tested, and hadIC50s of less than 100 nM.

Compounds disclosed herein are also active in inhibiting OVCAR8 cancercell proliferation. Compounds disclosed in Table 1 were synthesized andtested, and had IC50s of less than 100 nM.

Glycerol Lysis Buffer Recipe

Final Concentration  2 ml 1M Tris-HCl pH 7.4  20 mM  3 ml 5M NaCl 150 mM15 ml 100% glycerol 15%  1 ml 100% Triton-X 100  1% 79 ml DistilledWater (100 ml total)

To each 1 ml add before use: 100 ul 10% SDS, 5 μl 200 mM PMSF, 10 ulHALT Protease, 1 ul of Benzonase

Cell Viability or Cell Proliferation Assay

Cell culture media: All cells were grown in RPMI-1640 (Gibco11875-119)with 2 mM Glutamine+10% FBS (Gibco16000-044) unless otherwise noted.

Cells were plated in black, 96-well Corning Costar 3603 at densities toallow for Log phase growth throughout the experiment (see table below)on Day 0. Cells were allowed to settle and attach overnight. Cellsplated in triplicate for each concentration and control examined. On Day1, media was removed from cells and replaced with RPMI-1640+10% DialyzedFBS (Gibco26400-044) with indicated concentration of GLS-1 inhibitor(starting concentration 1 μM, diluted 1:3 to a final of 0.004 μM), 0.01%DMSO or 1 μM Staurosporine as indicated. Cells were incubated withcompound or relevant controls for 72 hours. On Day 4, 72 hours aftertreatment, cells were analyzed by Cell Titer Glo (PromegaG7573) permanufacturer's instructions. Plates were read in luminescence mode on aPheraStarFS plate reader. Data was collected, replicates were averaged,standard deviations calculated and analyzed through nonlinear regressionanalysis with four (4) parameters to generate IC50s for each cell linetested.

Cell Line Cells Plated Per Well (96-well) OVCAR8 2,500 TOV21G 2,000COV504 2,000 ES-2 1,750 MCAS 1,000 OV56 1,250 OAW-42 4,000 OVCAR4291,250 PA-1 1,000 OVCAR432 1,000 OVCAR420 2,000 OVCAR433 1,250 OVCAR51,000 OV7 4,000 SK-OV-3 1,250 OV90 7,500 OAW-28 4,000 EF027 1,250 OV17R5,000 IGROV1 2,000 FUOV1 7,000 EFO21 5,000 OVCAR4 3,750 A2780 4,000SW626 2,000

Analysis of Glutathione Levels: Cells were plated in 96-well CorningCostar 3603 at 10,000 cells/well on Day 0. Cells were allowed to settleand attach overnight. Cells plated in triplicate for each concentrationand control examined. On Day 1, media was removed from cells andreplaced with RPMI-1640+10% Dialyzed FBS (Gibco26400-044) with indicatedconcentration of Compound 1 (starting concentration luM, diluted 1:3 toa final of 0.004 uM), or 0.01% DMSO as indicated. Cells were incubatedwith compound or relevant controls for 24 hours. On Day 2, 24 hoursafter treatment, cells were analyzed by GSH-Glo (PromegaV6912) permanufacturer's instructions. Plates were read in luminescence mode on aPheraStarFS plate reader. Duplicate wells were plated and then stainedwith crystal violet in order to correct for differences in cell growthas described below.

GLS-1 Enzymatic Activity Assay

The following is a non-limiting example of an assay that may be used toevaluate the biological efficacy of compounds useful in the methods ofthe invention or to determine if a compound is a selective inhibitor ofGLS-1.

The inhibition of purified recombinant human GAC by varyingconcentrations of inhibitors may be assessed via a dual-coupledenzymatic assay. The glutamate produced by the glutaminase reaction isused by glutamate oxidase to produce α-ketoglutarate, ammonia, andhydrogen peroxide. This hydrogen peroxide is subsequently used byhorseradish peroxidase to produce resorufin in the presence of AmplexUltraRed. The assay buffer consisted of 50 mM HEPES (pH 7.4), 0.25 mMEDTA and 0.1 mM Triton X-100. GAC was incubated with potassium phosphate(10 minutes at room temperature) prior to incubation with inhibitor (10minutes at room temperature). The final reaction conditions were asfollows: 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamateoxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mL horseradishperoxidase (Sigma), 75 μM Amplex UltraRed (Life Technologies), and 1%(v/v) DMSO. The production of resorufin was monitored on a Perkin ElmerEnvision plate reader (excitation 530 nm, emission 590 nm) either in akinetics or endpoint mode (at 20 minutes). IC₅₀ values were calculatedusing a four-parameter logistic curve fit.

Target Engagement Assay

OVCA cells were plated in 96-well Corning Costar 3603 at 10,000cells/well on Day 0.24 h after plating, media was removed and replacedwith media containing 1 μM Compound 1 or 0.01% DMSO (control). At 24 h,100 μl of media was collected from each sample. Media was frozen at −80°C. after collection. One well for each condition was fixed and stainedwith crystal violet to normalize for cell number (see protocol below).All crystal violet samples were solubilized and used to normalize thecollected data. Collected samples were analyzed for glutamine andglutamate content using the Y512950 Bio-analyzer equipped with membranesthat measure the relevant chemistries.

Crystal Violet Staining Protocol

500 mg crystal violet powder was combined with 35 ml 100% EtOH insterile 50 ml tube until it dissolved. The mixture was transferred to a1 L beaker, the tube washed out with ddH₂O, and ddH₂O was added to thebeaker to 500 ml final volume. The mixture was filter sterilized using a0.45 μm filter. Media was removed and discarded (including wells withoutcells for background). 50 μL crystal violet stain+10% EtOH was added tothe wells and stained for 10 min. The stain was removed from the wellsand discarded into a waste container. Water was run in a large beaker orice bucket and the plate was repeatedly rinsed in the water. Theremaining liquid was shaken out into the sink. The plate was left upsidedown, uncovered for at least 5 hours to dry. Once completely dry, it wassolubilized in 10% acetic acid by adding 100 μL to all stained wells.After 5 minutes, all wells were pipet mixed and after another 5 minutesread on Pherastar at OD 590 nm.

DNA Damage Staining and Quantification

OVCAR8 and OVCAR420 cells were seeded in 96-well Corning Costar 3603plates and treated with 0.01% DMSO or 1 μM Compound 1 for 48 hours.Cells were then fixed using 4% Paraformaldehyde and permeabilized using0.5% TritonX100. Fixed cells were stained with Anti-γH2AX, rabbit(Bethyl IHC-00059) (1:500) for 2 h at 37 C. The secondary antibody usedwas Alexa Fluor 546 donkey anti-rabbit (Invitrogen A10040) (1:150)simultaneously with Hoechst 33342, Trihydrochloride, Trihydrate(Invitrogen, H3570) (1:1000) for 1 h at 37 C. DNA damage was measured asa γH2AX signal per nucleus using PerkinElmer High Content ScreeningSystem Operetta with the Harmony software. First, nuclei were selectedusing Hoechst staining. Then, single cells were gated based on nucleussize and shape. Apoptotic and mitotic cells were excluded based onmaximum Hoechst intensity in the nucleus. DNA damage foci wereidentified within the gated nuclei using the “find spots in Alexa 546channel” analysis block. Finally, the total intensity of Alexa 546within all “spots” in the nucleus, or the total number of spots in thenucleus, averaged per cell, was calculated.

Metabolomics Sample Preparation

OVCAR8 cells were plated at 3×10⁶ cells per 10 cm dish and OVCAR429,IGROV1, and OVCAR4 were plated at 3.5×10⁶ cells per 10 cm dish 24 hours(24 h) prior to treatment. Cells were treated on day zero (d.0) at timezero (t.0) with 0.01% DMSO, 1 μM, 100 nM, or 10 nM of Compound 1 for 24h. On day 1 (d.1), 2 hours (2 h) prior to harvest, samples were washedwith 5 mL media containing 10% dialyzed FBS. 10 mL of media was added toplates and plates were returned to the incubator for 2 h. Media was thenaspirated from plates and 4 mL of 80% chilled MeOH (−80° C.) wasimmediately added. Plates were incubated at −80° C. for 15 minutes.Cells were then scraped with cell scraper to release cells while keepingplates on dry ice. The MeOH/cell lysate mixture was collected andtransferred to conical tubes on dry ice. Tubes were centrifuged at fullspeed in 4° C. chilled centrifuge for 5 minutes. Supernatant wastransferred to another set of conical tubes on dry ice by decanting. Theremaining pellet was re-suspended in 500 μL of cold MeOH and the mixturewas moved to a microcentrifuge tube. Microfuge tubes were spun at fullspeed for 5 minutes in 4° C. chilled microcentrifuge. Supernatant wastransferred to conical tube with previously collected supernatant. Thisstep was repeated 2 times. 1 mL of collected supernatant was transferredto pre-labeled sample/submission tubes and submit for drying byspeedvac. Samples were then submitted to the Beth Israel DeaconessMedical Center Mass Spectrometry core facility for analysis by LC MS/MSusing an Agilent SCIEX 5500 QTRAP to profile 258 metabolites (Yuan, M.,et al. Nature Protocols 2012, 7(5) 872-81).

Metabolomics Sample Analysis

Differentially abundance analysis of metabolite levels was carried outusing a moderated t-test function from Bioconductor's limma package(Smyth 2005). A normalization factor was applied to the metabolite peakdata to account of variation in cell counts. The normalization factorwas calculated by first defining a vector, Nmed, to be the median peakarea for each metabolite across a set of samples. Next, for a sample i,a vector of fold-changes, FCi, is computed by dividing the metabolitepeak areas by Nmed. The scaling factor for sample i was set as themedian value of FCi. Significant changes in metabolite levels weredefined an abundance change of a least 1.5 fold increase/decrease andt-test p-value <=0.05. The symbols were assigned according to thep-values with the following cutoffs: +=p>0.005 and p<=0.05, *=p>0.00005and p<=0.005, **=p<=0.00005.

Gene Expression Analysis

Affymetrix U133 Plus2.0 microarrays were performed for each condition intriplicates. Robust multi-array average (RMA) method was used withdefault options (with background correction, quantile normalization, andlog transformation) to normalize raw data from batches usingR/Bioconductor's affy package (Irizarry et al 2003). Differentiallyexpression analysis was carried out using a moderated t-test functionfrom Bioconductor's limma package (Smyth 2005). A gene is called asdifferentially expressed if FDR corrected p-value is less than 0.05.

Analysis of Reactive Oxygen Species Production

One day prior to the experiment, OVCAR-8 and OVCAR420 cells were seededin 6 well dishes (250,000 cells per well). The day of the experiment,cells were treated with 0.01% DMSO or 1 μM Compound 1 for 24 hours. Thefollowing day, an allocated well was treated with hydrogen peroxide for20-30 minutes as a positive control for ROS generation. All wells werethen stained with CM-H2DCFDA (5 μM, Life Technologies) for 30 minutes at37° C., washed with 1×PBS, harvested by trypsinization, and re-suspendedin 400 μl phenol-free RPMI medium. Cells were strained through a 40 μMcell strainer and analyzed using a flow cytometer (LSR Fortessa). Valuesdisplayed are fold changes compared to DMSO for mean fluorescenceintensity of entire population.

EXAMPLES

Several embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1. HGSOC Cell Lines are Differentially Sensitive to GLSInhibition

To investigate the role of GLS in regulating HGSOC cell proliferation wefirst characterized a chemical probe designed to specifically inhibitGLS-1. We have confirmed that this tool compound (compound 1) is apotent inhibitor of GLS-1 (IC₅₀ 7.5 nM) in a coupled in vitro enzymaticassay (FIG. 1(a)). The inhibition of GAC by varying concentrations ofcompound 1 was assessed using a dual-coupled enzymatic assay (pH 7.4)containing 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamateoxidase, 1 mM glutamine, 100 mU/mL HRP and 75 μM Amplex Ultra Red. Theproduction of resorufin was monitored on a Perkin Elmer Envision platereader (excitation 530 nm, emission 590 nm) for 20 min.

In cells, the chemical probe inhibits the conversion of glutamine toglutamate; a measure of GLS-dependent activity (FIGS. 1(b)-(e)). Targetengagement can be measured after treatment with compound 1. As a measureof compound activity on GLS in cells, the conversion of glutamine toglutamate was measured in extracellular media samples treated with DMSO(control) or compound 1. Treatment of cells for 24 hours with compound 1prevented the conversion of glutamine to glutamate (as measured by theratio of glutamate to glutamine). This inhibition was evident by adecrease in the GLU:GLN ratio in cell lysates.

Interestingly, while all cell lines tested showed a decrease in GLSactivity, only some cells showed an anti-proliferative response afterGLS1i treatment (FIG. 2(a)-(b)). Differential response to GLS-1inhibition is observed in OVCA lines. OVCAR8, OVCAR429, OVCAR4, andIGROV1 cell lines were treated with compound 1 and 72 hour viability wasanalyzed. OVCAR8 and OVCAR429 show nM sensitivity to GLS1i, and OVCAR4and IGROV1 cells are resistant to treatment with GLS1i.

The potential therapeutic benefit of GLS-1 inhibition in HGSOC was alsoinvestigated. Through a cell screening campaign (conducted as describedabove in the Cell Viability or Cell Proliferation Assay), asubpopulation of HGSOC cell lines that are hypersensitive to GLS-1inhibition (IC₅₀ 2D viability ≤50 nM in “responder”; >10 μM in“non-responder”) was identified. FIG. 4 shows a waterfall plot depictingdifferential response to GLS-1 inhibition in broad panel of OVCA lines:(a) OVCAR8; (b) TOV21G; (c) COV504; (d) ES-2; (e) MCAS; (f) OV56; (g)OAW-42; (h) OVCAR429; (i) PA-1; (j) OVCAR432; (k) OVCAR420; (1)OVCAR433; (m) OVCAR5; (n) OV7; (o) SKOV3; (p) OV90; (q) OAW-28; (r)EF027; (s) OV17R; (t) IGROV-1; (u) FUOV-1; (v) EFO21; (w) OVCAR4; (x)A2780; (y) SW626. These cell lines were treated with GLS1i in a doseresponse and viability was analyzed after 72 hours. A subset of OVCAlines show low nM sensitivity to GLS1i while others do not show anyresponse. Interestingly, nearly all lines (responders andnon-responders) show an acute depletion of intracellular glutamatelevels indicative of glutaminase inhibition. This suggests that a subsetof HGSOC cell lines have a specific dependence on glutamine metabolismfor survival.

Example 2. GLS Inhibition Alters Redox Balance in Responder Cell Lines

To interrogate the role of glutaminase in the regulation of tumormetabolism, targeted liquid chromatography-tandem mass spectrometry(LC-MS/MS) metabolomic studies were performed to comprehensivelycharacterize metabolic alterations following GLS-1 inhibition (FIGS. 5and 6) Metabolites quantified are: (TCA) (a) fumarate; (b) maleate; (c)oxaloacetate; (d) citrate; (e) citrate-isocitrate; (f) isocitrate; (g)alpha-ketoglutarate; (h) succinate; (glutamate synthesis) (i) alanine;(j) aspartate; (purines) (k) AICA ribonucleotide (AICAR); (l) inosinemonophosphate (IMP); (m) inosine; (n) hypoxanthine; (pentose phosphate)(o) ribose 5-phosphate (R5P); (p) sedoheptulose-7-phosphate (S7P); (q)sedoheptulose-1,7-bisphosphate (SBP); (r) glyceraldehyde 3-phosphate(GAP); (s) dihydroxyacetone phosphate (DHAP); (t) erythrose 4-phosphate(E4P). This analysis revealed that GLS-1 inhibition in “responder” celllines induced significant metabolic changes involving multiple pathways,the most significant of which are intermediates in the TCA cycle.

Consistent with the role of glutamine in regulating intracellular redoxbalance, GLS-1 inhibition decreased the effective concentration ofglutathione, and induced activity of the pentose phosphate and ribosesalvage pathways. Interestingly, changes in these metabolic pathwayswere muted in “non-responder” HGSOC lines, confirming a differentialaddiction to glutamine. However, one common response across cell lineswas activation of metabolic pathways designed to replenish glutamatepools in cells through aspartate and alanine metabolism, suggesting apossible compensatory mechanism sufficient for the survival of“non-responder” cell lines when challenged with GLS-1 inhibition.

OVCAR8, OVCAR429, OVCAR4, and IGROV1 cells treated with 1 μM compound 1for 24 hours were subjected to global metabolic profiling afterisolation of lysates by methanol extraction. Changes in metabolitelevels reflected an accumulation of glutamine in cells after GLS-1inhibition accompanied by a decrease in TCA cycle activity; likely dueto decreased glutamine anaplerosis. Additionally, decreases in freenucleotide pools were seen. These decreased metabolic activities wereaccompanied by an increase in pentose phosphate pathway activity,presumably to attempt to maintain redox balance in the absence ofglutathione derived from the GLS-dependent breakdown of glutamine toglutamate, and activation of a ribose salvage pathway in response tocompound 1 treatment, suggesting that cells are attempting to maintainnucleotide and thiol pools in response to decreased glutaminolysis. Itshould be noted that these metabolic changes are muted or absent innon-responder lines treated with compound 1. Interestingly, severalmetabolites which serve as alternative starting points for glutamatesynthesis were decreased across all samples. This is possibly analternative mechanism to produce glutamate void of the glutamine toglutamate conversion by GLS.

Example 3. GLS Inhibition Induces ROS and DNA Damage in OVCA Cells

GLS-dependence is driven by addiction to glutamine-dependent,glutathione-mediated redox maintenance in OVCA responder cell lines.Metabolic profiling coupled with the differential sensitivity of HGSOCcell lines (see Metabolomics Sample Preparation and Analysis protocolsdescribed above) suggested an important role for glutamine-derivedglutathione in regulating redox balance. Consistent with thishypothesis, GLS-1 inhibition induced a significant decrease inintracellular glutathione and a concurrent increase in reactive oxygenspecies (FIGS. 7 and 8) in responder lines. Glutathione levels aredecreased after treatment with compound 1 in responder cell lines—GSHlevels were analyzed in cell lines after treatment with compound 1. Asseen in FIG. 7, GSH levels were significantly decreased in OVCAR420,OVCAR429, and OVCAR8 cells after 24 hours of treatment (1 μM). Arepresentative non-responder line, OVCAR4, does not show a significantdecrease in GSH levels after GLS-1 inhibition, suggesting that thesecells have alternate mechanisms for maintaining redox balance.

As seen in FIG. 8, loss of GSH after GLS-1 inhibition leads to anaccumulation of intracellular reactive oxygen species (ROS). Cells wereanalyzed for accumulation of intracellular ROS after 48 h treatment withcompound 1 by staining with CM-H₂DCFDA (an indicator of generaloxidative stress) and analysis by flow cytometry. For each condition,calculation of mean fluorescence intensity allows for the comparison ofROS accumulation between control and treated samples. Shown areresponder cells: (a) OVCAR420; (b) OVCAR429, with GFP-A on the x-axis,and count on the y-axis. Treatment of responder cells with compound 1 (1μM) for 48 hours induces an accumulation of ROS that correlates with thedepletion of the GSH pool from cells.

GLS-1 inhibition causes cell cycle arrest. Cells were analyzed for cellcycle state using a standard BrdU incorporation assay. As seen in FIGS.9 and 10, after 24 hours of treatment with GLS1i, responder cell linesshowed an accumulation of cells in G1, with a sever depletion of cellsundergoing active DNA replication in S-phase.

GLS-1 inhibition induces oxidative stress that leads to accumulation ofDNA damage. HGSOC cells treated with either DMSO or compound 1 for 48hours were stained with Hoechst's Stain (nuclei) and an antibody toγH2AX (DNA damage) and then analyzed by high-content imaging. γH2AX fociwere counted and quantified per nuclei. As seen in FIG. 11,quantification (bar plot shown) reveals that treatment with compound 1induces DNA damage in multiple responder cell lines. Moreover, weobserved an accumulation of γ2AX foci, a marker of DNA damage, and thelikely cause of anti-proliferative effects observed upon GLS-1inhibition (FIG. 11).

Application of exogenous GSH to responder cell lines rescues compound1-induced proliferation defects. OVCAR420 and OVCAR429 cells weretreated with compound 1 at the indicated doses in a 72 h growth assay.At the same time, a subset of the cells treated with compound 1 receivedapplications of cell-permeable GSH (once at the time of compound 1treatment, once 24 h post-treatment and once 48 h post-treatment).Application of exogenous GSH rescued a significant portion of cellgrowth that was inhibited after treatment with compound 1. GLS1i-inducedinhibition of cell proliferation were rescued upon treatment withexogenous glutathione (FIG. 12). Of note, neither glutathione depletion,ROS induction, nor an accumulation of DNA damage was observed innon-responder cell lines. This suggests that a subpopulation of HGSOC issensitive to GLS-1 inhibitors due to a dependence on glutamine-derivedglutathione for maintenance of redox balance. Concurrently, themetabolic profiles of non-responder lines suggested alternativemechanisms for maintaining redox balance.

Example 4. High ASNS Expression Provides an Alternative Source ofGlutamate and Confers Resistance to GLS Inhibition

Next, molecular insight into the differential response to GLS inhibitionwas sought. Given the observed metabolic shift towards pathways involvedin replenishing pools of glutamate, attention was focused onglutamate-producing enzymes (FIG. 13) which might enable non-responderlines to maintain glutamate pools in the absence of GLS activity andconsequently prevent depletion of glutathione.

Enzymes including asparagine synthetase (ASNS) which converts aspartateto asparagine and glutamate, as well as glutamic pyruvate transaminase(GPT2) which converts alpha-ketoglutarate to pyruvate and glutamate wereinterrogated.

Differential expression of glutamate producing enzymes was observed inresponder and non-responder cell lines. OVCAR8, OVCAR429, OVCAR4,OVCAR420, and IGROV1 cells treated with 1 μM of compound 1 for 24 hourswere subjected to gene profiling after RNA isolation. Data analysisrevealed that several glutamate producing enzymes such as asparaginesynthetase (ASNS) and glutamic pyruvate transaminase (GPT2), had higherexpression in OVCA non-responder cell lines compared to responder celllines. This finding correlates with metabolomics profiling data thatshowed changes in metabolites that are involved in glutamate synthesis.

Data from cell line profiling using reverse phase protein array (RPPA)or western blotting showed increased expression of ASNS in non-respondercell lines compared to responder lines (FIGS. 14(a) and (b),respectively). FIG. 14 depicts RPPA protein expression analysis in OVCAcell lines. ASNS protein expression was compared across a subset ofresponder and non-responder cell lines. Higher ASNS levels were observedin non-responder cell lines (unpublished data).

FIG. 15 shows Western blot of ASNS expression across a panel of OVCAcell lines. Cell lines screened are (a) A2780; (b) OVCAR4; (c) SW626;(d) OV56; (e) OVCAR420; (f) SKOV3 (g) OVCAR5; (h) OVCAR8; (i) OCAR429;(j) OAW-28; (k) FUOV1; (1) OVCAR3; (m) OAW-42; (n) TOV21G; (o) COV504;(p) OVCAR433; (q) OVCAR432; (r) OV7. ASNS is differentially expressed inOVCA cell lines. Cells that do not respond to GLS1i have higher ASNSexpression compared to responder cell lines. These increased levels ofASNS likely enable cells to cope with GLS-1 inhibition by circumventingthe requirement for glutamine-derived glutamate to maintain glutathionepools. These tumors are able to maintain glutathione levels throughaspartate-derived glutamate, and would consequently be insensitive toGLS-1 inhibition.

Indeed, over-expression of ASNS in OVCAR8 cells, which normally expresslittle to no ASNS, confers resistance to GLS1i (FIG. 16). OVCAR8 cellswhich respond to GLS1i and express no ASNS (FIG. 15) were transducedwith virus encoding an ASNS construct (ASNS OE—cells treated withnon-diluted viral supernatant, ASNS OE 1:10 or ASNS OE 1:100—cellstreated with diluted viral supernatant, 1:10 or 1:100, respectively) andanalyzed by western blot (FIGS. 16(a) and (b)). Cells over-expressingASNS display resistance to compound 1 in a 72 hour proliferation assay(FIG. 16(c)). Taken together, these data suggest that high ASNSexpression may act as a negative predictor of response to GLS1i.

Example 5. Immuno-Histochemical Staining can Determine ASNS Levels andAct as a Predictor of Response

Immuno-histochemical (IHC) staining for ASNS confirms differentialexpression between responders and non-responders. In order tointerrogate the feasibility of detecting ASNS expression levels intumors, an IHC assay for ASNS was established. OVCAR4 cells, whichexpress high levels of ASNS (FIGS. 14 and 15) were treated with a poolof siRNA directed at ASNS. After conditions were established to providea significant decrease in ASNS expression (FIG. 17), cytoblocks of thesecells were prepared and analyzed by IHC with several antibodies. FIG. 17shows the confirmation of the knockdown of ASNS protein. OVCAR4 cellswere transiently transfected with ASNS siRNA and non-targeting control(NTC) for 7 hours. After 72 hours, cells were lysed and proteinisolated. ASNS knock-down efficiency was analyzed by western blot.Results show a significant decrease in ASNS protein expression afterASNS siRNA transfection.

An ASNS expression assay was optimized (FIG. 17 (c) and (d)) andanalysis of a panel of responder and non-responder cell linesillustrates that high ASNS scores are a negative predicator of responsein HGSOC cell lines (FIG. 18). FIG. 17 (c) and (d) shows ASNSimmunohistochemical antibody validation. An aliquot of transfected cellsused in FIG. 17, were collected, fixed, and processed forimmunohistochemical staining. Results show a significant decrease inASNS expression after ASNS siRNA transfection.

FIG. 18 shows that an IHC assay for ASNS expression can stratifyresponse to GLS-1 inhibition. OVCAR420, OVCAR429, OVCAR4, and A2780(FIGS. 18 (a)-(d), respectively) were cultured normally and collected.Cell pellets were coded for blinded analysis and fixed into cytoblocks.Samples were prepared and analyzed by a blinded pathologist and scoredaccording to ASNS expression. OVCAR429 (compound 1 IC50=76 nM) and OVCAR420 (compound 1 IC50=59 nM) received scores of—and −/+ respectively on a4 point graded scale (−, +, ++, +++). Thus, these cell lines werecorrectly predicted to respond to GLS-1 inhibition. Conversely, OVCAR4(no response to compound 1) and A2780 (no response to compound 1) bothreceived a blinded score of +++, indicating high ASNS expression andpredicting no response to GLS-1 inhibition.

FIG. 19 shows that the tissue microarrays from ovarian cancer patientsillustrate actionable ASNS-low populations in patients who arerefractory to frontline therapies. Tissue microarrays collected from 134ovarian cancer patients who had undergone frontline therapies includingplatinum-based treatments (Cisplatin/Carbotaxol/etc.) were stained forexpression of ASNS. Example stains of TMA cores are shown in the boxesillustrating low versus high ASNS expression. A summary of scoring fromthese cores reveals 24% of patients with no ASNS expression, and another44% with medium levels of ASNS expression. Both populations would bepredicted to respond to GLS-1 inhibition.

Example 6. Glutaminase Inhibition Inhibits Tumor Growth in ASNS_(low)Models of Ovarian Cancer

As seen in FIG. 20(a), OVCAR-8 subcutaneous xenografts are sensitive toGLS-1 inhibition. OVCAR-8 cells were implanted subcutaneously intoathymic mice and allowed to grow into an established tumor. Animals werethen treated with compound 2, for three weeks (100 mpk, twice a day) byoral gavage. Treatment with compound 2 resulted in a significant tumorgrowth inhibition after three weeks of dosing.

FIG. 20(b) shows that glutaminase inhibition inhibits tumor progressionin an orthotopic model of ovarian cancer. SK-OV-3 cells were injectedinto the intraperitoneal cavity of athymic nude mice and allow to growfor 7 days. At day 7, animals were treated with the in vivo toolcompound to inhibit GLS-1 and animals were monitored for clinicaldisease signs. At day 21, vehicle treated animals began to exhibit signsof disease burden and all groups were sacrificed and tumor nodule weightwas analyzed. Single-agent-treatment with compound 2 showed a robustreduction in combined nodule weight per animal compared to vehicle orpaclitaxel alone (a current standard of care agent). Additionally, thecombination of compound 2 and paclitaxel showed some added benefit whencompared to compound 2 treatment alone.

As seen in FIG. 21, OVCAR-8 tumors demonstrate altered glutaminemetabolism after GLS-1 inhibition. OVCAR-8 tumors were dosed for 1, 7 or21 days and then harvested from animals. Tumor lysates were analyzed bymass spec for the presence of glutamine and glutamate. Tumors treatedwith GLS1i showed decreased ratios of glutamate to glutamine over thecourse of the three week experiment, confirming on-target activity oftool compounds.

FIG. 22 shows that glutaminase inhibition decreases proliferation invivo. SK-OV-3 cells were implanted subcutaneously into athymic nude miceand animals were treated with compound 1 for 3 days. Tumors wereharvested and then stained for phospho-histone H3, a marker of cellproliferation. Representative images are shown, along with a blindedpathologist's score indicating that treatment with single-agentglutaminase inhibitor decrease proliferation in vivo.

Example 7. Inhibition of Glutaminolysis and Tumor Growth in the PatientDerived Tumor Models

While ASNS expression levels stratify responder and non-responder celllines, we asked whether or not this would be an actionable patientpopulation in the clinic and if patient-derived pre-clinical modelswould respond to GLSi. Consequently, we measured the expression of ASNSin tumor tissue isolated from patient-derived xenograft models of HGSOCvia IHC.

Tissue sections collected from 25 ovarian cancer patients who hadundergone frontline therapies including platinum-based treatments(cisplatin/carbotaxol/etc.) were stained for expression of ASNS,evaluated by a trained pathologist and graded on a 4-point scale withlongitudinal sections graded by H&E staining for ovarian cancersubtyping via histopathological features (FIG. 23(a)). The first model,scored as a 0, was not included in further study.

1 OV15398  9 OV5392 17 OV14702 2 OV15577 10 OV15631 18 OV14871 3 OV529711 OV15841 19 OV14972 4 OV5304 12 OV5296 20 OV15123 5 OV5308 13 OV530921 OV15209 6 OV5383 14 OV5390 22 OV15612 7 OV5385 15 OV13950 23 OV156968 OV5387 16 OV13951 24 OV5397

Of these 24 remaining models, approximately sections 40% were ASNS-low(FIG. 24(a)). One specific model, OV5392, was verified as high-gradeserous subtype and selected for a follow-up in vivo study.

The the anti-tumor effects of Compound 2 were tested in vivo in anASNS^(low) HGSOC patient-derived xenograft (PDX) model using human tumortissue that was identified through immunohistochemistry to be ASNS^(low)and characterized through histological phenotyping to be of a high-gradeserous subtype, implanted in to NOD/SCID mice.

Compound 2 inhibits tumor growth of an ASNS^(low) PDX xenograft as asingle agent in NOD/SCID mice (FIG. 23(b)). In this experiment, Compound2, administered orally at a dose of 100 mg/kg, BID for 39 days inhibitedtumor growth (TGI=50%, p<0.0001 for tumor volumes after treatment withCompound 2 vs vehicle control). These data illustrate that Compound 2 isable to inhibit glutaminolysis and tumor growth in the patient derivedtumor models, and may have significant additional benefit incombinations with standard of care chemotherapy.

Example 8. Glutaminase Inhibitors Exhibit Anti-Proliferative Effects onCells with Low Levels of ASNS

As seen in FIG. 25, tumor cells are able to cope with oxidative stressduring conditions of rapid proliferation by maintaining high levels ofglutathione (Tumor Cell, top panel). When GLS-1 is inhibited in a subsetof these cells (Treated Tumor Cell-Responder, middle panel),intracellular pools of glutamate are drastically reduced, and thus,glutathione synthesis is inhibited. This response shifts the redoxbalance of tumor cells causing an increase in ROS accumulation and DNAdamage leading to cell death. In certain cases, even though GLS-1 isinhibited, tumor cells with high ASNS expression are still able toproduce intracellular glutamate by converting aspartate to glutamate andasparagine. Consequently, these tumor cells are able to maintain redoxhomeostasis (Treated Tumor Cell-Non-responder, bottom panel).

This demonstrates that ASNS is a negative indicator of response to GLS-1inhibition (FIG. 25) and a clinical assay to examine ASNS expression inpre-treatment biopsies could be used to stratify patients for GLS-1inhibitor treatment in the clinic.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present disclosure. However, thedisclosure described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the disclosure. Anyequivalent embodiments are intended to be within the scope of thisdisclosure. Indeed, various modifications of the disclosure in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description, which do not departfrom the spirit or scope of the present inventive discovery. Suchmodifications are also intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A method of treating cancer in a subject whosecancer cells express low levels of asparagine synthetase (ASNS), asdefined by an Histophathology Score (H-score) of less than or equal to100 by immunohistochemical staining, comprising administering aglutaminase (GLS) inhibitor to said subject.
 2. The method of claim 1wherein the GLS inhibitor is selective for glutaminase-1 (GLS-1).
 3. Themethod of claim 2, wherein the cancer is chosen from bladder cancer,bone marrow cancer, breast cancer, cancer of the central nervous system,cervical cancer, colon cancer, endometrial cancer, cancer of the gastricsystem, head and neck cancer, kidney cancer, leukemia, liver cancer,lung cancer, lymphoma, muscle cancer, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, thyroid cancer, or a variant thereof. 4.The method of claim 3, wherein the cancer is chosen from ovarian, skin,liver, prostate, breast, colon, lung, head and neck cancers or alymphoma.
 5. The method of claim 4, wherein the cancer is ovariancancer.
 6. The method of claim 5, wherein the ovarian cancer ishigh-grade serous ovarian cancer (HGSOC).
 7. The method of claim 6,wherein the ovarian cancer is nonresectable or relapsed HGSOC.
 8. Themethod of claim 5, further comprising administering anotherpharmaceutically active compound.
 9. The method of claim 8, wherein saidanother pharmaceutically active compound is chosen from carboplatin,cisplatin, paclitaxel, and docetaxel.
 10. The method of claim 9, whereinsaid another pharmaceutically active compound is chosen from carboplatinand cisplatin.
 11. The method of claim 9, wherein said anotherpharmaceutically active compound is chosen from paclitaxel anddocetaxel.
 12. The method of claim 11, wherein said anotherpharmaceutically active compound is paclitaxel.
 13. The method of claim2, wherein the GLS-1 inhibitor binds an allosteric pocket on thesolvent-exposed region of the GLS-1 dimer in the binding pocket presentin the vicinity of Leu321, Phe322, Leu323, and Tyr394 from bothmonomers.
 14. The method of claim 2, wherein the GLS-1 inhibitor hasFormula I:

or a salt thereof, wherein: n is chosen from 3, 4, and 5; each R^(x) andR^(y) is independently chosen from alkyl, cyano, H, and halo, whereintwo R^(x) groups together with the atoms to which they are attachedoptionally form a cycloalkyl ring; A¹ and A² are independently chosenfrom C—H, C—F, and N; R¹ and R⁴ are independently chosen from alkenyl,alkoxy, alkyl, aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H,halo, haloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl,heterocycloalkylalkyl, hydroxyl, C(R³)₂C(O)R³, C(R³)₂C(O)N(R³)₂,C(R³)₂N(R³)₂, C(R³)₂NR³C(O)R³, C(R³)₂NR³C(O)OR³, C(R³)₂NR³C(O)N(R³)₂,C(R³)₂NR³S(O)R³, C(R³)₂NR³S(O)₂R³, N(R³)₂, NR³C(O)R³, NR³C(O)OR³,NR³C(O)N(R³)₂, NR³S(O)R³, NR³S(O)₂R³, C(O)N(R³)₂, S(O)N(R³)₂,S(O)₂N(R³)₂, C(O)R³, SR³, S(O)R³, and S(O)₂R³, wherein each R¹ and R⁴may be optionally substituted with between 0 and 3 R^(z) groups; R² ischosen from alkyl, heterocycloalkyl, cyano, cycloalkyl, H, halo, andhaloalkyl, wherein R¹ and R² together with the atoms to which they areattached optionally form an form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups; each R³ is independently chosen from alkenyl,alkoxy, alkyl, aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H,halo, haloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl,heterocycloalkylalkyl, and hydroxyl, wherein each R³ may be optionallysubstituted with between 0 and 3 R^(z) groups, wherein two R³ groupstogether with the atoms to which they are attached optionally form anaryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups; each R^(Z)group is independently chosen from alkenyl, alkoxy, alkoxyalkyl,alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl, alkoxycycloalkylalkyl,alkoxyhaloalkyl, alkoxyheteroaryl, alkoxyheteroarylalkyl,alkoxyheterocycloalkyl, alkoxyheterocycloalkylalkyl, alkyl, alkylaryl,alkylarylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁶)₂,NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂, NR⁶S(O)C(R⁶)₃,NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂, C(O)C(R⁶)₃,SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; each R⁶ is independently chosenfrom alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, and hydroxyl, wherein two R⁶groups together with the atoms to which they are attached optionallyform an aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring, whichmay be optionally substituted with between 0 and 3 R^(x) groups; and Zis heteroaryl, which may be optionally substituted.
 15. The method ofclaim 2, wherein the GLS-1 inhibitor has Formula II:

or a salt thereof, wherein: n is chosen from 3, 4, and 5; each R^(x) andR^(y) is independently chosen from alkyl, cyano, H, and halo, whereintwo R^(x) groups together with the atoms to which they are attachedoptionally form a cycloalkyl ring; A¹ and A² are independently chosenfrom N and CH; A³ is chosen from N and CR²; R¹ is chosen from alkenyl,alkyl, aryl, arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo,haloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl,heterocycloalkylalkyl, C(O)N(R³)₂, and C(O)C(R³)₃, wherein R¹ may beoptionally substituted with between 0 and 3 R^(z) groups, R² is chosenfrom alkenyl, alkoxy, alkyl, aryl, arylalkyl, cyano, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, heterocycloalkylalkyl, hydroxyl, C(O)N(R³)₂,C(O)C(R³)₃, C(O)OH, C(O)OC(R³)₃, wherein R¹ and R² together with theatoms to which they are attached optionally form an form an aryl,cycloalkyl, heteroaryl, or heterocycloalkyl ring, which may beoptionally substituted with between 0 and 3 R^(z) groups; each R³ isindependently chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl,cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, and hydroxyl,wherein each R³ may be optionally substituted with between 0 and 3 R^(z)groups, wherein two R³ groups together with the atoms to which they areattached optionally form an aryl, cycloalkyl, heteroaryl, orheterocycloalkyl ring, which may be optionally substituted with between0 and 3 R^(z) groups; R⁴ is chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, N(R³)₂, NR³C(O)C(R³)₃, NR³C(O)OC(R³)₃, NR³C(O)N(R³)₂,NR³S(O)C(R³)₃, NR³S(O)₂C(R³)₃, C(O)N(R³)₂, S(O)N(R³)₂, S(O)₂N(R³)₂,C(O)C(R³)₃, SC(R³)₃, S(O)C(R³)₃, and S(O)₂C(R³)₃, wherein R⁴ may beoptionally substituted with between 0 and 3 R^(z) groups, each R^(z)group is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, halo, haloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl,hydroxyl, oxo, N(R⁶)₂, NR⁶C(O)C(R⁶)₃, NR⁶C(O)OC(R⁶)₃, NR⁶C(O)N(R⁶)₂,NR⁶S(O)C(R⁶)₃, NR⁶S(O)₂C(R⁶)₃, C(O)N(R⁶)₂, S(O)N(R⁶)₂, S(O)₂N(R⁶)₂,C(O)C(R⁶)₃, SC(R⁶)₃, S(O)C(R⁶)₃, and S(O)₂C(R⁶)₃; each R⁶ isindependently chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl,cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, and hydroxyl,wherein two R⁶ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with between 0 and 3 R^(x)groups; and Z is heteroaryl, which may be optionally substituted. 16.The method of claim 2, wherein the GLS-1 inhibitor has Formula Mc:

or a salt thereof, wherein: R^(X) is chosen from fluoro and H; R¹ ischosen from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl,cycloalkylalkyl, H, halo, haloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, and heterocycloalkylalkyl, wherein R¹ may beoptionally substituted with one to three R^(Z) groups; each R⁴ isindependently chosen from alkenyl, alkoxy, alkyl, aryl, arylalkyl,cyano, cycloalkyl, cycloalkylalkyl, H, halo, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl, and hydroxyl,wherein R⁴ may be optionally substituted with one to three R^(Z) groups;each R^(Z) group is independently chosen from alkenyl, alkoxy,alkoxyalkyl, alkoxyaryl, alkoxyarylalkyl, alkoxycycloalkyl,alkoxycycloalkylalkyl, alkoxyhaloalkyl, alkoxyheteroaryl,alkoxyheteroarylalkyl, alkoxyheterocycloalkyl,alkoxyheterocycloalkylalkyl, alkyl, alkylaryl, alkylarylalkyl,alkylcycloalkyl, alkylcycloalkylalkyl, alkylheteroaryl,alkylheteroarylalkyl, alkylheterocycloalkyl, alkylheterocycloalkylalkyl,aryl, arylalkyl, arylalkyloxy, arylhaloalkyl, aryloxy, cyano,cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, cycloalkylhaloalkyl,cycloalkyloxy, H, halo, haloalkoxy, haloalkoxyalkyl, haloalkoxyaryl,haloalkoxyarylalkyl, haloalkoxycycloalkyl, haloalkoxycycloalkylalkyl,haloalkoxyheteroaryl, haloalkoxyheteroarylalkyl,haloalkoxyheterocycloalkyl, haloalkoxyheterocycloalkylalkyl, haloalkyl,haloalkylaryl, haloalkylarylalkyl, haloalkylcycloalkyl,haloalkylcycloalkylalkyl, haloalkylheteroaryl, haloalkylheteroarylalkyl,haloalkylheterocycloalkyl, haloalkylheterocycloalkylalkyl, haloaryl,haloarylalkyl, haloarylalkyloxy, haloaryloxy, halocycloalkyl,halocycloalkylalkyl, halocycloalkylalkyloxy, halocycloalkyloxy,haloheteroaryl, haloheteroarylalkyl, haloheteroarylalkyloxy,haloheteroaryloxy, haloheterocycloalkyl, haloheterocycloalkylalkyl,haloheterocycloalkylalkyloxy, haloheterocycloalkyloxy, heteroaryl,heteroarylalkyl, heteroarylalkyloxy, heteroarylhaloalkyl, heteroaryloxy,heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkyloxy,heterocycloalkylhaloalkyl, heterocycloalkyloxy, hydroxyl, oxo, N(R⁵)₂,NR⁵C(O)R⁵, NR⁵C(O)OR⁵, NR⁵C(O)N(R⁵)₂, NR⁵S(O)R⁵, NR⁵S(O)₂R⁵, C(O)N(R⁵)₂,S(O)N(R⁵)₂, S(O)₂N(R⁵)₂, C(O)R⁵, C(O)OR⁵, SR⁵, S(O)R⁵, and S(O)₂R⁵; andeach R⁵ is independently chosen from alkenyl, alkoxy, alkyl, aryl,arylalkyl, cyano, cycloalkyl, cycloalkylalkyl, H, haloalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereintwo R⁵ groups together with the atoms to which they are attachedoptionally form an aryl, cycloalkyl, heteroaryl, or heterocycloalkylring, which may be optionally substituted with one to three R^(X)groups.
 17. The method of claim 2, wherein the GLS-1 inhibitor isselected from:

or a salt thereof.
 18. The method of claim 2, wherein the GLS-1inhibitor is selected from: a)(S)-2-hydroxy-2-phenyl-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide,b)N,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide),also known as BPTES, c)2-(pyridin-2-yl)-N-{5-[4-(6-{2-[3-(trifluoromethoxy)phenyl]acetamido}pyridazin-3-yl)butyl]-1,3,4-thiadiazol-2-yl}acetamide,also known as CB-839, d)N,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide),e)N-methyl-1-{4-[6-(2-{4-[3-(trifluoromethoxy)phenyl]pyridin-2-yl}acetamido)pyridazin-3-yl]butyl}-1H-1,2,3-triazole-4-carboxamide,f)1-(2-fluoro-4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1H-1,2,3-triazole-4-carboxamide,g)1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,h)N-(pyridin-2-ylmethyl)-5-(3-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide,i)(R)-1-(2-fluoro-4-(6-(2-(4-(3-(trifluoromethoxy)phenyl)pyridin-2-yl)acetamido)-pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,j)(R)-1-(2-fluoro-4-(6-(2-(4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,k)(R)-1-(2-fluoro-4-(6-(2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,l)(R)-1-(4-(6-(2-(4-(cyclopropyldifluoromethyl)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,m)(R)-1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,n)(R)-1-(2-fluoro-4-(6-(2-(1-(3-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)-acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,o)1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,p)1-(4-(6-(2-(4-cyclobutoxypyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,q)1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)butyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,r)1-(4-(6-(2-(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,s)(R)-1-(4-(6-(2-(4-cyclopropylpyridin-2-yl)acetamido)pyridazin-3-yl)-2-fluorobutyl)-N-methyl-1H-1,2,3-triazole-4-carboxamide,t)5-(3-(6-(2-(pyridin-2-yl)acetamido)pyridazin-3-yl)pyrrolidin-1-yl)-N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide,and u)N,N′-(5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(both or either of 1S,3S and 1R,2R enantiomers), or a salt thereof. 19.The method of claim 2, wherein the GLS-1 inhibitor is

or a salt thereof.
 20. The method of claim 2, wherein the subject ishuman.
 21. The method of claim 2, further comprising administeringanother pharmaceutically active compound.
 22. The method of claim 21,wherein the other pharmaceutically active compound is an anti-canceragent.
 23. The method of claim 22, wherein the anti-cancer agent ischosen from a platinum-based agent, a taxane-based agent, animmunotherapy, an immuno-oncotherapy, and a targeted therapy.
 24. Themethod of claim 23, wherein the targeted therapy is an inhibitor of MEKkinase, HSP90, CDK4, or the mTOR pathway.
 25. The method of claim 24,wherein the method further comprises administering non-chemical methodsof cancer treatment.
 26. The method of claim 25, wherein the methodfurther comprises administering radiation therapy.
 27. The method ofclaim 25, wherein the method further comprises administering surgery,thermoablation, focused ultrasound therapy, cryotherapy, or anycombination thereof.